Leucine-based motif and clostridial neurotoxins

ABSTRACT

Modified neurotoxin comprising neurotoxin including structural modification, wherein the structural modification alters the biological persistence, such as the biological half-life and/or a biological activity of the modified neurotoxin relative to an identical neurotoxin without the structural modification. In one embodiment, methods of making the modified neurotoxin include using recombinant techniques. In another embodiment, methods of using the modified neurotoxin to treat conditions include treating various disorders, neuromuscular aliments and pain.

CROSS REFERENCE

[0001] This application is a continuation in part of application Ser.No. 09/620,840, filed Jul. 21, 2000.

BACKGROUND

[0002] The present invention relates to modified neurotoxins,particularly modified Clostridial neurotoxins, and use thereof to treatvarious conditions including conditions that have been treated usingnaturally occurring botulinum toxins.

[0003] Botulinum toxin, for example, botulinum toxin type A, has beenused in the treatment of numerous conditions including pain, skeletalmuscle conditions, smooth muscle conditions and glandular conditions.Botulinum toxins are also used for cosmetic purposes.

[0004] Numerous examples exist for treatment using botulinum toxin. Forexamples of treating pain see Aoki, et al., U.S. Pat. No. 6,113,915 andAoki, et al., U.S. Pat. No. 5,721,215. For an example of treating aneuromuscular disorder, see U.S. Pat. No. 5,053,005, which suggeststreating curvature of the juvenile spine, i.e., scoliosis, with anacetylcholine release inhibitor, preferably botulinum toxin A. For thetreatment of strabismus with botulinum toxin type A, see Elston, J. S.,et al., British Journal of Ophthalmology, 1985, 69, 718-724 and 891-896.For the treatment of blepharospasm with botulinum toxin type A, seeAdenis, J. P., et al., J. Fr. Ophthalmol., 1990, 13 (5) at pages259-264. For treating spasmodic and oromandibular dystonia torticollis,see Jankovic et al., Neurology, 1987, 37, 616-623. Spasmodic dysphoniahas also been treated with botulinum toxin type A. See Blitzer et al.,Ann. Otol. Rhino. Laryngol, 1985, 94, 591-594. Lingual dystonia wastreated with botulinum toxin type A according to Brin et al., Adv.Neurol. (1987) 50, 599-608. Cohen et al., Neurology (1987) 37 (Suppl.1), 123-4, discloses the treatment of writer's cramp with botulinumtoxin type A.

[0005] It would be beneficial to have botulinum toxins with alteredbiological persistence and/or altered biological activity. For example,a botulinum toxin can be used to immobilize muscles and prevent limbmovements after tendon surgery to facilitate recovery. It would bebeneficial to have a botulinum toxin (such as a botulinum toxin type A)which exhibits a reduced period of biological persistence so that apatient can regain muscle use and mobility at about the time theyrecover from surgery. Furthermore, a botulinum toxin with an alteredbiological activity, such as an enhanced biological activity can haveutility as a more efficient toxin (i.e. more potent per unit amount oftoxin), so that less toxin can be used.

[0006] Additionally, there is a need for modified neurotoxins (such asmodified Clostridial toxins) which can exhibit an enhanced period ofbiological persistence and modified neurotoxins (such as modifiedClostridial toxins) with reduced biological persistence and/orbiological activity and methods for preparing such toxins.

Definitions

[0007] Before proceeding to describe the present invention, thefollowing definitions are provided and apply herein.

[0008] “Heavy chain” means the heavy chain of a Clostridial neurotoxin.It has a molecular weight of about 100 kDa and can be referred to hereinas Heavy chain or as H.

[0009] “H_(N)” means a fragment (having a molecular weight of about 50kDa) derived from the Heavy chain of a Clostridial neurotoxin which isapproximately equivalent to the amino terminal segment of the Heavychain, or the portion corresponding to that fragment in the intact Heavychain. It is believed to contain the portion of the natural or wild typeClostridial neurotoxin involved in the translocation of the light chainacross an intracellular endosomal membrane.

[0010] “H_(C)” means a fragment (about 50 kDa) derived from the Heavychain of a Clostridial neurotoxin which is approximately equivalent tothe carboxyl terminal segment of the Heavy chain, or the portioncorresponding to that fragment in the intact Heavy chain. It is believedto be immunogenic and to contain the portion of the natural or wild typeClostridial neurotoxin involved in high affinity binding to variousneurons (including motor neurons), and other types of target cells.

[0011] “Light chain” means the light chain of a Clostridial neurotoxin.It has a molecular weight of about 50 kDa, and can be referred to aslight chain, L or as the proteolytic domain (amino acid sequence) of aClostridial neurotoxin. The light chain is believed to be effective asan inhibitor of exocytosis, including as an inhibitor ofneurotransmitter (i.e. acetylcholine) release when the light chain ispresent in the cytoplasm of a target cell.

[0012] “Neurotoxin” means a molecule that is capable of interfering withthe functions of a cell, including a neuron. The “neurotoxin” can benaturally occurring or man-made. The interfered with function can beexocytosis.

[0013] “Modified neurotoxin” means a neurotoxin which includes astructural modification. In other words, a “modified neurotoxin” is aneurotoxin which has been modified by a structural modification. Thestructural modification changes the biological persistence, such as thebiological half-life (i.e. the duration of action of the neurotoxin)and/or the biological activity of the modified neurotoxin relative tothe neurotoxin from which the modified neurotoxin is made or derived.The modified neurotoxin is structurally different from a naturallyexisting neurotoxin.

[0014] “Mutation” means a structural modification of a naturallyoccurring protein or nucleic acid sequence. For example, in the case ofnucleic acid mutations, a mutation can be a deletion, addition orsubstitution of one or more nucleotides in the DNA sequence. In the caseof a protein sequence mutation, the mutation can be a deletion, additionor substitution of one or more amino acids in a protein sequence. Forexample, a specific amino acid comprising a protein sequence can besubstituted for another amino acid, for example, an amino acid selectedfrom a group which includes the amino acids alanine, aspargine,cysteine, aspartic acid, glutamic acid, phenylalanine, glycine,histidine, isoleucine, lysine, leucine, methionine, proline, glutamine,arginine, serine, threonine, valine, tryptophan, tyrosine or any othernatural or non-naturally occurring amino acid or chemically modifiedamino acids. Mutations to a protein sequence can be the result ofmutations to DNA sequences that when transcribed, and the resulting mRNAtranslated, produce the mutated protein sequence. Mutations to a proteinsequence can also be created by fusing a peptide sequence containing thedesired mutation to a desired protein sequence.

[0015] “Structural modification” means any change to a neurotoxin thatmakes it physically or chemically different from an identical neurotoxinwithout the structural modification.

[0016] “Biological persistence” or “persistence” means the time durationof interference or influence caused by a neurotoxin or a modifiedneurotoxin with a cellular (such as a neuronal) function, including thetemporal duration of an inhibition of exocytosis (such as exocytosis ofneurotransmitter, for example, acetylcholine) from a cell, such as aneuron.

[0017] “Biological half-life” or “half-life” means the time that theconcentration of a neurotoxin or a modified neurotoxin, preferably theactive portion of the neurotoxin or modified neurotoxin, for example,the light chain of Clostridial toxins, is reduced to half of theoriginal concentration in a mammalian cell, such as in a mammalianneuron.

[0018] “Biological activity” or “activity” means the amount of cellularexocytosis inhibited from a cell per unit of time, such as exocytosis ofa neurotransmitter from a neuron.

[0019] “Target cell” means a cell (including a neuron) with a bindingaffinity for a neurotoxin or for a modified neurotoxin.

SUMMARY

[0020] New structurally modified neurotoxins have been discovered. Thepresent structurally modified neurotoxins can provide substantialbenefits, for example, enhanced or decreased biological persistenceand/or biological half-life and/or enhanced or decreased biologicalactivity as compared to the unmodified neurotoxin.

[0021] In accordance with the present invention, there are providedstructurally modified neurotoxins, which include a neurotoxin and astructural modification. The structural modification is effective toalter a biological persistence of the structurally modified neurotoxinrelative to an identical neurotoxin without the structural modification.Also, the structurally modified neurotoxin is structurally differentfrom a naturally existing neurotoxin.

[0022] The present invention also encompasses a modified neurotoxincomprising a neurotoxin with a structural modification, wherein saidstructural modification is effective to alter a biological activity ofsaid modified neurotoxin relative to an identical neurotoxin withoutsaid structural modification, and wherein said modified neurotoxin isstructurally different from a naturally existing neurotoxin. Thisstructural modification can be effective to reduce an exocytosis from atarget cell by more than the amount of the exocytosis reduced from thetarget cell by an identical neurotoxin without said structuralmodification. Alternately, the structural modification can be effectiveto reduce an exocytosis from a target cell by less than the amount ofthe exocytosis reduced from the cell by an identical neurotoxin withoutsaid structural modification. Significantly, the exocytosis can beexocytosis of a neurotransmitter and the modified neurotoxin can exhibitan altered biological activity without exhibiting an altered biologicalpersistence. The structural modification can comprise a leucine-basedmotif. Additionally, the modified neurotoxin can exhibits an alteredbiological activity as well as an altered biological persistence. Thepresent invention also includes the circumstances where: (a) themodified neurotoxin exhibits an increased biological activity as well asan increased biological persistence; (b) the modified neurotoxinexhibits an increased biological activity and a reduced biologicalpersistence; (c) the modified neurotoxin exhibits a decreased biologicalactivity and a decreased biological persistence, and; (d) the modifiedneurotoxin exhibits an decreased biological activity and an increasedbiological persistence.

[0023] Importantly, a unit amount (i.e. on a molar basis) of themodified neurotoxin can be more efficient to reduce an exocytosis from acell than is a unit amount of the naturally existing neurotoxin. Inother words, a unit amount of a modified neurotoxin, such as a modifiedbotulinum toxin type A, can cleave its' intracellular substrate (SNAP)in a manner such that a greater inhibition of neurotransmitterexocytosis results (i.e. less neurotransmitter is released from thecell), as compared to the inhibition of neurotransmitter exocytosisexhibited by the naturally occurring neurotoxin.

[0024] Further in accordance with the present invention, arestructurally modified neurotoxins, wherein a structural modification iseffective to enhance a biological persistence of the modifiedneurotoxin. The enhanced biological persistence of the structurallymodified neurotoxin can be due, at least in part, to an increasedhalf-life and/or biological activity of the structurally modifiedneurotoxin.

[0025] Still further in accordance with the present invention, there areprovided structurally modified neurotoxins wherein a biologicalpersistence of the structurally modified neurotoxin is reduced relativeto that of an identical neurotoxin without the structural modification.This reduction in biological persistence can be due, at least in part,to a decreased biological half-life and/or activity of the structurallymodified neurotoxins.

[0026] Still further in accordance with the present invention, there areprovided structurally modified neurotoxins wherein the structuralmodification comprises a number of amino acids. For example, the numberof amino acids comprising the structural modification can be 1 or moreamino acids, from 1 to about 22 amino acids, from 2 to about 10 aminoacids, and from about 4 to about 7 amino acids.

[0027] In one embodiment, the structural modifications of thestructurally modified neurotoxins can comprise an amino acid. The aminoacid can comprise an R group containing a number of carbons. Forexample, the number of carbon atoms in the amino acid can be 1 or more,from 1 to about 20 carbons, from 1 to about 12 carbons, from 1 to about9 carbons, from 2 to about 6 carbons, and about 4 carbons. R group asused in this application refers to amino acid side chains. For example,the R group for alanine is CH₃, and, for example, the R group for serineis CH₂OH.

[0028] In another embodiment, there are provided structurally modifiedneurotoxins wherein the modification comprises an amino acid. The aminoacid can comprise an R group which is substantially hydrocarbyl.

[0029] In still another embodiment, there are provided structurallymodified neurotoxins wherein the structural modification comprises anamino acid. The amino acid further can comprise an R group that includesat least one heteroatom.

[0030] Further in accordance with the present invention, there areprovided structurally modified neurotoxins wherein the structuralmodification comprises, for example, a leucine-based motif, atyrosine-based motif, and/or an amino acid derivative. Examples of anamino acid derivative that can comprise a structurally modifiedneurotoxin are a myristylated amino acid, an N-glycosylated amino acid,and a phosphorylated amino acid. The phosphorylated amino acids can bephosphorylated by, for example, casein kinase II, protein kinase C, andtyrosine kinase.

[0031] Still further in accordance with the present invention, there areprovided structurally modified neurotoxins which can include astructural modification. The neurotoxin can comprise three amino acidsequence regions. The first region can be effective as a cellularbinding moiety. This binding moiety can be a binding moiety for a targetcell, such as a neuron. The binding moiety can be the carboxyl terminusof a botulinum toxin heavy chain. It is well known that the carboxylterminus of a botulinum toxin heavy chain can be effective to bind, forexample, receptors found on certain cells, including certain nervecells. In one embodiment, the carboxyl terminus binds to receptors foundon a presynaptic membrane of a nerve cell. The second region can beeffective to translocate a structurally modified neurotoxin, or a partof a structurally modified neurotoxin across an endosome membrane. Thethird region can be effective to inhibit exocytosis from a target cell.The inhibition of exocytosis can be inhibition of neurotransmitterrelease, such as acetylcholine from a presynaptic membrane. For example,it is well known that the botulinum toxin light chain is effective toinhibit, for example, acetylcholine (as well as other neurotransmitters)release from various neuronal and non-neuronal cells.

[0032] At least one of the first, second or third regions can besubstantially derived from a Clostridial neurotoxin. The third regioncan include the structural modification. In addition, the modifiedneurotoxin can be structurally different from a naturally existingneurotoxin. Also, the structural modification can be effective to altera biological persistence of the modified neurotoxin relative to anidentical neurotoxin without the structural modification.

[0033] In one embodiment, there are provided structurally modifiedneurotoxins, wherein the neurotoxin can be botulinum serotype A, B, C₁,C₂, D, E, F, G, tetanus toxin and/or mixtures thereof.

[0034] In another embodiment, there are provided structurally modifiedneurotoxins where the third region can be derived from botulinum toxinserotype A. In addition, there are provided structurally modifiedneurotoxins wherein the third region can not be derived from botulinumserotype A.

[0035] In still another embodiment, there are provided structurallymodified neurotoxins wherein the structural modification includes abiological persistence enhancing component effective to enhance thebiological persistence of the structurally modified neurotoxin. Theenhancing of the biological persistence can be at least in part due toan increase in biological half-life and/or activity of the structurallymodified neurotoxin.

[0036] Further in accordance with the present invention, there areprovided structurally modified neurotoxins comprising a biologicalpersistence enhancing component, wherein the biological persistenceenhancing component can comprise a leucine-based motif. Theleucine-based motif can comprise a run of 7 amino acids, where a quintetof amino acids and a duplet of amino acids can comprise theleucine-based motif. The quintet of amino acids can define the aminoterminal end of the leucine-based motif. The duplet of amino acids candefine the carboxyl end of the leucine-based motif. There are providedstructurally modified neurotoxins wherein the quintet of amino acids cancomprise one or more acidic amino acids. For example, the acidic aminoacid can be glutamate or aspartate. The quintet of amino acids cancomprise a hydroxyl containing amino acid. The hydroxyl containing aminoacid can be, for example, a serine, a threonine or a tyrosine. Thishydroxyl containing amino acid can be phosphorylated. At least one aminoacid comprising the duplet of amino acids can be a leucine, isoleucine,methionine, alanine, phenylalanine, tryptophan, valine or tyrosine. Inaddition, the duplet of amino acids in the leucine-based motif can beleucine-leucine, leucine-isoleucine, isoleucine-leucine orisoleucine-isoleucine, leucine-methionine. The leucine-based motif canbe an amino acid sequence ofphenylalanine-glutamate-phenylalanine-tyrosine-lysine-leucine-leucine.

[0037] In one embodiment, there are provided structurally modifiedneurotoxins wherein the modification can be a tyrosine-based motif. Thetyrosine-based motif can comprise four amino acids. The amino acid atthe N-terminal end of the tyrosine-based motif can be a tyrosine. Theamino acid at the C-terminal end of the tyrosine-based motif can be ahydrophobic amino acid.

[0038] Further in accordance with the present invention, the thirdregion can be derived from botulinum toxin serotype A or form one of theother botulinum toxin serotypes.

[0039] Still further in accordance with the present invention, there areprovided structurally modified neurotoxins where the biologicalpersistence of the structurally modified neurotoxin can be reducedrelative to an identical neurotoxin without the structural modification.The reduced biological persistence can be in part due a decreasedbiological half-life and/or to a decrease biological activity of theneurotoxin.

[0040] In one embodiment, there are provided structurally modifiedneurotoxins, where the structural modification can include aleucine-based motif with a mutation of one or more amino acidscomprising the leucine-based motif. The mutation can be a deletion orsubstitution of one or more amino acids of the leucine-based motif.

[0041] In another embodiment, there are provided structurally modifiedneurotoxins, where the structural modification includes a tyrosine-basedmotif with a mutation of one or more amino acids comprising thetyrosine-based motif. For example, the mutation can be a deletion orsubstitution of one or more amino acids of the tyrosine-based motif.

[0042] In still another embodiment, there are provided structurallymodified neurotoxins, wherein the structural modification comprises anamino acid derivative with a mutation of the amino acid derivative or amutation to a nucleotide or amino acid sequence which codes for thederivativization of the amino acid. For example, a deletion orsubstitution of the derivatized amino acid or a nucleotide or amino acidsequence responsible for a derivatization of the derivatized amino acid.The amino acid derivative can be, for example, a myristylated aminoacid, an N-glycosylated amino acid, or a phosphorylated amino acid. Thephosphorylated amino acid can be produced by, for example, casein kinaseII, protein kinase C or tyrosine kinase.

[0043] In one embodiment of the present invention, there are providedstructurally modified neurotoxins, wherein the first, second and/orthird regions of the structurally modified neurotoxins can be producedby recombinant DNA methodologies, i.e. produced recombinantly.

[0044] In another embodiment of the present invention, there areprovided structurally modified neurotoxins, wherein the first, secondand/or third region of the neurotoxin is isolated from a naturallyexisting Clostridial neurotoxin.

[0045] Another embodiment of the present invention provides a modifiedneurotoxin comprising a botulinum toxin (such as a botulinum toxin typeA) which includes a structural modification which is effective to altera biological persistence of the modified neurotoxin relative to anidentical neurotoxin without the structural modification. The structuralmodification can comprise a deletion of amino acids 416 to 437 from alight chain of the neurotoxin (FIG. 3).

[0046] In still another embodiment of the present invention there isprovided a modified neurotoxin (such as a botulinum toxin type A) whichincludes a structural modification which is effective to alter abiological persistence of the modified neurotoxin relative to anidentical neurotoxin without the structural modification. The structuralmodification can comprise a deletion of amino acids 1 to 8 from a lightchain of the neurotoxin (FIG. 3).

[0047] Still further in accordance with the present invention there isprovided a modified neurotoxin, such as a botulinum toxin type A, whichincludes a structural modification which is effective to alter abiological persistence of the modified neurotoxin relative to anidentical neurotoxin without the structural modification. The structuralmodification can comprise a deletion of amino acids 1 to 8 and 416 to437 from a light chain of the neurotoxin (FIG. 3).

[0048] Still further in accordance with the present invention, there isprovided a modified botulinum toxin, such as a modified botulinum toxintype A, which includes a structural modification effective to alter abiological persistence of the modified neurotoxin relative to anidentical neurotoxin without said structural modification. Thestructural modification can comprise a substitution of leucine atposition 427 for an alanine and a substitution of leucine at position428 for an alanine in a light chain of said neurotoxin (FIG. 3).

[0049] Additionally, the scope of the present invention also includesmethods for enhancing the biological persistence and/or or for enhancingthe biological activity of a neurotoxin. In these methods, a structuralmodification can be fused or added to the neurotoxin, for example, thestructural modification can be a biological persistence enhancingcomponent and/or a biological activity enhancing component. Examples ofstructural modifications that can be fused or added to the neurotoxinare a leucine-based motif, a tyrosine-based motif and an amino acidderivative. Examples of amino acid derivatives are a myristylated aminoacid, an N-glycosylated amino acid, and a phosphorylated amino acid. Anamino acid can be phosphorylated by, for example, protein kinase C,caseine kinase II or tyrosine kinase.

[0050] Also in accordance with the present invention, there are providedmethods for reducing the biological persistence and/or for reducing thebiological activity of a neurotoxin. These methods can comprise a stepof mutating an amino acid of the neurotoxin. For example, an amino acidof a leucine-based motif within the neurotoxin can be mutated. Also, forexample, one or more amino acids within a tyrosine-based motif of theneurotoxin can be mutated. Also, for example, an amino acid derivativefor DNA or amino acid sequence responsible for the derivatization of theamino acid can be mutated. The derivatized amino acid can be amyristylated amino acid, a N-glycosylated amino acid, or aphosphorylated amino acid. The phosphorylated amino acid can be producedby, for example, protein kinase C, caseine kinase II and tyrosinekinase. These mutations can be, for example, amino acid deletions oramino acids substitutions.

[0051] The present invention also includes methods for treating acondition. The methods can comprise a step of administering an effectivedose of a structurally modified neurotoxin to a mammal to treat acondition. The structurally modified neurotoxin can include a structuralmodification. The structural modification is effective to alter thebiological persistence and/or the biological activity of the neurotoxin.These methods for treating a condition can utilize a neurotoxin thatdoes not comprise a leucine-based motif. Also, these methods fortreating a condition can utilize a neurotoxin, which includes abiological persistence enhancing component and/or a biological activityenhancing component. The biological persistence or activity enhancingcomponent can comprise, for example, a tyrosine-based motif, aleucine-based motif or an amino acid derivative. The amino acidderivative can be, for example, a myristylated amino acid, anN-glycosylated amino acid or a phosphorylated amino acid. Thephosphorylated amino acid can be produced by, for example, proteinkinase C, caseine kinase II or tyrosine kinase. The condition treatedcan be a neuromuscular disorder, an autonomic disorder or pain. Thetreatment of a neuromuscular disorder can comprise a step of locallyadministering an effective amount of a modified neurotoxin to a muscleor a group of muscles. A method for treating an autonomic disorder cancomprise a step of locally administering an effective amount of amodified neurotoxin to a gland or glands. A method for treating pain cancomprise a step of administering an effective amount of a modifiedneurotoxin to the site of the pain. In addition, the treatment of paincan comprise a step of administering an effective amount of a modifiedneurotoxin to the spinal cord.

[0052] Still further in accordance with the present invention, there areprovided methods for treating with modified neurotoxins conditionsincluding spasmodic dysphonia, laryngeal dystonia, oromandibulardysphonia, lingual dystonia, cervical dystonia, focal hand dystonia,blepharospasm, strabismus, hemifacial spasm, eyelid disorder, cerebralpalsy, focal spasticity, spasmodic colitis, neurogenic bladder, anismus,limb spasticity, tics, tremors, bruxism, anal fissure, achalasia,dysphagia, lacrimation, hyperhydrosis, excessive salivation, excessivegastrointestinal secretions, pain from muscle spasms, headache pain,brow furrows and skin wrinkles.

BRIEF DESCRIPTION OF THE DRAWINGS

[0053]FIG. 1 shows localization of GFP-botulinum toxin A light chain in(nerve growth factor) NGF-differentiated live PC12 cells visualized on afluorescence inverted microscope.

[0054]FIG. 2 shows the localization of GFP-truncated botulinum toxin Alight chain in NGF-differentiated live PC12 cells visualized on afluorescence inverted microscope.

[0055]FIG. 3 shows the amino acid sequence for botulinum type A lightchain. The amino acid sequence shown, minus the underlined amino acidsrepresents botulinum type A truncated light chain.

[0056]FIG. 4 shows the localization of GFP-botulinum toxin A light chainwith LL to AA mutation at position 427 and 428 in NGF-differentiatedlive PC12 cells visualized on a fluorescence inverted microscope.

[0057]FIG. 5 shows localization of fluorescently labeled anti-SNAP-25visualized in horizontal confocal sections ofstaurosporine-differentiated PC12 cells.

[0058]FIG. 6 shows an X-ray crystalographic structure of botulinum toxintype A.

[0059]FIG. 7 shows localization of GFP-botulinum type B neurotoxin lightchain in NGF-differentiated live PC12 cells visualized on a fluorescenceinverted microscope.

[0060]FIG. 8 shows sequence alignment and consensus sequence forbotulinum toxin type A HallA light chain and botulinum toxin type BDanish I light chain.

[0061]FIG. 9 is a graph which illustrates the results of an in vitroELISA assay carried out by the inventors demonstrating that a truncatedLC/A in vitro cleaves substrate at a slower rate or less efficientlythan does non-truncated LC/A.

[0062]FIG. 10 shows a comparison of LC/A constructs expressed from E.coli for in vitro analysis.

DETAILED DESCRIPTION

[0063] The present invention is based upon the discovery that thebiological persistence and/or the biological activity of a neurotoxincan be altered by structurally modifying the neurotoxin. In other words,a modified neurotoxin with an altered biological persistence and/orbiological activity can be formed from a neurotoxin containing orincluding a structural modification. In one embodiment, the structuralmodification includes the fusing of a biological persistence enhancingcomponent to the primary structure of a neurotoxin to enhance itsbiological persistence. In a preferred embodiment, the biologicalpersistence enhancing component is a leucine-based motif. Even morepreferably, the biological half-life and/or the biological activity ofthe modified neurotoxin is enhanced by about 100%. Generally speaking,the modified neurotoxin has a biological persistence of about 20% to300% more than an identical neurotoxin without the structuralmodification. That is, for example, the modified neurotoxin includingthe biological persistence enhancing component is able to cause asubstantial inhibition of neurotransmitter release for example,acetylcholine from a nerve terminal for about 20% to about 300% longerthan a neurotoxin that is not modified.

[0064] The present invention also includes within its scope a modifiedneurotoxin with a biological activity altered as compared to thebiological activity of the native or unmodified neurotoxin. For example,the modified neurotoxin can exhibit a reduced or an enhanced inhibitionof exocytosis (such as exocytosis of a neurotransmitter) from a targetcell with or without any alteration in the biological persistence of themodified neurotoxin.

[0065] In a broad embodiment of the present invention, a leucine-basedmotif is a run of seven amino acids. The run is organized into twogroups. The first five amino acids starting from the amino terminal ofthe leucine-based motif form a “quintet of amino acids.” The two aminoacids immediately following the quintet of amino acids form a “duplet ofamino acids.” In a preferred embodiment, the duplet of amino acids islocated at the carboxyl terminal region of the leucine-based motif. Inanother preferred embodiment, the quintet of amino acids includes atleast one acidic amino acid selected from a group consisting of aglutamate and an aspartate.

[0066] The duplet of amino acid includes at least one hydrophobic aminoacid, for example leucine, isoleucine, methionine, alanine,phenylalanine, tryptophan, valine or tyrosine. Preferably, the duplet ofamino acid is a leucine-leucine, a leucine-isoleucine, anisoleucine-leucine or an isoleucine-isoleucine, leucine-methionine. Evenmore preferably, the duplet is a leucine-leucine.

[0067] In one embodiment, the leucine-based motif is xDxxxLL, wherein xcan be any amino acids. In another embodiment, the leucine-based motifis xExxxLL, wherein E is glutamic acid. In another embodiment, theduplet of amino acids can include an isoleucine or a methionine, formingxDxxxLI or xDxxxLM, respectively. Additionally, the aspartic acid, D,can be replaced by a glutamic acid, E, to form xExxxLI, xExxxIL andxExxxLM. In a preferred embodiment, the leucine-based motif isphenylalanine-glutamate-phenylalanine-tyrosine-lysine- leucine-leucine,SEQID #1.

[0068] In another embodiment, the quintet of amino acids comprises atleast one hydroxyl containing amino acid, for example, a serine, athreonine or a tyrosine. Preferably, the hydroxyl containing amino acidcan be phosphorylated. More preferably, the hydroxyl containing aminoacid is a serine which can be phosphorylated to allow for the binding ofadapter proteins.

[0069] Although non-modified amino acids are provided as examples, amodified amino acid is also contemplated to be within the scope of thisinvention. For example, leucine-based motif can include a halogenated,preferably, fluorinated leucine.

[0070] Various leucine-based motif are found in various species. A listof possible leucine-based motif derived from the various species thatcan be used in accordance with this invention is shown in Table 1. Thislist is not intended to be limiting. TABLE 1 Species Sequence SEQID#Botulinum type A FEFYKLL 1 Rat VMAT1 EEKRAIL 2 Rat VMAT 2 EEKMAIL 3 RatVAChT SERDVLL 4 Rat δ VDTQVLL 5 Mouse δ AEVQALL 6 Frog γ/δ SDKQNLL 7Chicken γ/δ SDRQNLI 8 Sheep δ ADTQVLM 9 Human CD3γ SDKQTLL 10 Human CD4SQIKRLL 11 Human δ ADTQALL 12 S. cerevisiae Vam3p NEQSPLL 13

[0071] VMAT is vesicular monoamine transporter; VACht is vesicularacetylcholine transporter and S. cerevisiae Vam3p is a yeast homologueof synaptobrevin. Italicized serine residues are potential sites ofphosphorylation.

[0072] The modified neurotoxin can be formed from any neurotoxin. Also,the modified neurotoxin can be formed from a fragment of a neurotoxin,for example, a botulinum toxin with a portion of the light chain and/orheavy chain removed. Preferably, the neurotoxin used is a Clostridialneurotoxin. A Clostridial neurotoxin comprises a polypeptide havingthree amino acid sequence regions. The first amino acid sequence regioncan include a target cell (i.e. a neuron) binding moiety which issubstantially completely derived from a neurotoxin selected from a groupconsisting of beratti toxin; butyricum toxin; tetanus toxin; botulinumtype A, B, C₁, D, E, F, and G. Preferably, the first amino acid sequenceregion is derived from the carboxyl terminal region of a toxin heavychain, H_(C). Also, the first amino acid sequence region can comprise atargeting moiety which can comprise a molecule (such as an amino acidsequence) that can bind to a receptor, such as a cell surface protein orother biological component on a target cell.

[0073] The second amino acid sequence region is effective to translocatethe polypeptide or a part thereof across an endosome membrane into thecytoplasm of a neuron. In one embodiment, the second amino acid sequenceregion of the polypeptide comprises an amine terminal of a heavy chain,HN, derived from a neurotoxin selected from a group consisting ofberatti toxin; butyricum toxin; tetanus toxin; botulinum type A, B, C₁,D, E, F, and G.

[0074] The third amino acid sequence region has therapeutic activitywhen it is released into the cytoplasm of a target cell, such as aneuron. In one embodiment, the third amino acid sequence region of thepolypeptide comprises a toxin light chain, L, derived from a neurotoxinselected from a group consisting of beratti toxin; butyricum toxin;tetanus toxin; botulinum type A, B, C₁, D, E, F, and G.

[0075] The Clostridial neurotoxin can be a hybrid neurotoxin. Forexample, each of the neurotoxin's amino acid sequence regions can bederived from a different Clostridial neurotoxin serotype. For example,in one embodiment, the polypeptide comprises a first amino acid sequenceregion derived from the H_(C) of the tetanus toxin, a second amino acidsequence region derived from the H_(N) of botulinum type B, and a thirdamino acid sequence region derived from the light chain of botulinumserotype E. All other possible combinations are included within thescope of the present invention.

[0076] Alternatively, all three of the amino acid sequence regions ofthe Clostridial neurotoxin can be from the same species and sameserotype. If all three amino acid sequence regions of the neurotoxin arefrom the same Clostridial neurotoxin species and serotype, theneurotoxin will be referred to by the species and serotype name. Forexample, a neurotoxin polypeptide can have its first, second and thirdamino acid sequence regions derived from Botulinum type E. In whichcase, the neurotoxin is referred as Botulinum type E.

[0077] Additionally, each of the three amino acid sequence regions canbe modified from the naturally occurring sequence from which they arederived. For example, the amino acid sequence region can have at leastone or more amino acids added or deleted as compared to the naturallyoccurring sequence.

[0078] A biological persistence enhancing component or a biologicalactivity enhancing component, for example a leucine-based motif, can befused with any of the above described neurotoxins to form a modifiedneurotoxin with an enhanced biological persistence and/or an enhancedbiological activity. “Fusing” as used in the context of this inventionincludes covalently adding to or covalently inserting in between aprimary structure of a neurotoxin. For example, a biological persistenceenhancing component and/or a biological activity enhancing component canbe added to a Clostridial neurotoxin which does not have a leucine-basedmotif in its primary structure. In one embodiment, a leucine-based motifis fused with a hybrid neurotoxin, wherein the third amino acid sequenceis derived from botulinum serotype A, B, C₁, C₂, D, E, F, or G. Inanother embodiment, the leucine-based motif is fused with a botulinumtype E.

[0079] In another embodiment, a biological persistence enhancingcomponent and/or a biological activity enhancing component is added to aneurotoxin by altering a cloned DNA sequence encoding the neurotoxin.For example, a DNA sequence encoding a biological persistence enhancingcomponent and/or a biological activity enhancing component is added to acloned DNA sequence encoding the neurotoxin into which the biologicalpersistence enhancing component and/or a biological activity enhancingcomponent is to be added. This can be done in a number of ways which arefamiliar to a molecular biologist of ordinary skill. For example, sitedirected mutagenesis or PCR cloning can be used to produce the desiredchange to the neurotoxin encoding DNA sequence. The DNA sequence canthen be reintroduced into a native host strain. In the case of botulinumtoxins the native host strain would be a Clostridium botulinum strain.Preferably, this host strain will be lacking the native botulinum toxingene. In an alternative method, the altered DNA can be introduced into aheterologous host system such as E. coli or other prokaryotes, yeast,insect cell lines or mammalian cell lines. Once the altered DNA has beenintroduced into its host, the recombinant toxin containing the addedbiological persistence enhancing component and/or a biological activityenhancing component can be produced by, for example, standardfermentation methodologies.

[0080] Similarly, a biological persistence enhancing component can beremoved from a neurotoxin. For example, site directed mutagenesis can beused to eliminate biological persistence enhancing components, forexample, a leucine-based motif.

[0081] Standard molecular biology techniques that can be used toaccomplish these and other genetic manipulations are found in Sambrooket al. (1989) which is incorporated in its entirety herein by reference.

[0082] In one embodiment, the leucine-based motif is fused with, oradded to, the third amino acid sequence region of the neurotoxin. In apreferred embodiment, the leucine-based motif is fused with, or addedto, the region towards the carboxylic terminal of the third amino acidsequence region. More preferably, the leucine-based motif is fused with,or added to, the carboxylic terminal of the third region of aneurotoxin. Even more preferably, the leucine-based motif is fused with,or added to the carboxylic terminal of the third region of botulinumtype E. The third amino acid sequence to which the leucine-based motifis fused or added can be a component of a hybrid or chimeric modifiedneurotoxin. For example, the leucine-based motif can be fused to oradded to the third amino acid sequence region (or a part thereof) of onebotulinum toxin type (i.e. a botulinum toxin type A), where theleucine-based motif-third amino acid sequence region has itself beenfused to or conjugated to first and second amino acid sequence regionsfrom another type (or types) of a botulinum toxin (such as botulinumtoxin type B and/or E).

[0083] In another embodiment, a structural modification of a neurotoxinwhich has a pre-existing biological persistence enhancing componentand/or a biological activity enhancing component, for example, aleucine-based motif includes deleting or substituting one or more aminoacids of the leucine-based motif. In addition, a modified neurotoxinincludes a structural modification which results in a neurotoxin withone or more amino acids deleted or substituted in the leucine-basedmotif. The removal or substitution of one or more amino acids from thepreexisting leucine-based motif is effective to reduce the biologicalpersistence and/or a biological activity of a modified neurotoxin. Forexample, the deletion or substitution of one or more amino acids of theleucine-based motif of botulinum type A reduces the biological half-lifeand/or the biological activity of the modified neurotoxin.

[0084] Amino acids that can be substituted for amino acids contained ina biological persistence enhancing component include alanine, aspargine,cysteine, aspartic acid, glutamic acid, phenylalanine, glycine,histidine, isoleucine, lysine, leucine, methionine, proline, glutamine,arginine, serine, threonine, valine, tryptophan, tyrosine and othernaturally occurring amino acids as well as non-standard amino acids.

[0085] In the present invention the native botulinum type A light chainhas been shown to localize to differentiated PC12 cell membranes in acharacteristic pattern. Biological persistence enhancing components areshown to substantially contribute to this localization.

[0086] The data of the present invention demonstrates that when thebotulinum toxin type A light chain is truncated or when theleucine-based motif is mutated, the light chain substantially loses itsability to localize to the membrane in its characteristic pattern.Localization to the cellular membrane is believed to be a key factor indetermining the biological persistence and/or the biological activity ofa botulinum toxin. This is because localization to a cell membrane canprotect the localized protein from inter-cellular protein degrading.

[0087]FIGS. 1 and 2 show that deletion of the leucine-based motif fromthe light chain of botulinum type A can change membrane localization ofthe type A light chain. FIG. 1 shows localization of GFP-light chain Afusion protein in differentiated PC12 cells. The GFP fusion proteinswere produced and visualized in differentiated PC12 cells using methodswell known to those skilled in the art, for example, as described inGalli et al (1998) Mol Biol Cell 9:1437-1448, incorporated in itsentirety herein by reference; also, for example, as described inMartinez-Arca et al (2000) J Cell Biol 149:889-899, also incorporated inits entirety herein by reference. Localization of a GFP-truncated lightchain A is shown in FIG. 2. Comparing FIGS. 1 and 2, it can be seen thatthe pattern of localization is completely altered by the deletion of theN-terminus and C-terminus comprising the leucine-based motif. FIG. 3shows the amino acid sequence of the botulinum type A light chain. Theunderlined amino acid sequences indicate the amino acids that weredeleted in the truncated mutant. The leucine-based motif is indicated bythe asterisked bracket.

[0088] Further studies have been done in the present invention toanalyze the effect of specific amino acid substitutions within theleucine-based motif. For example, in one study both leucine residuescontained in the leucine-based motif were substituted for alanineresidues. FIG. 4 shows the fluorescent image of differentiated PC12cells transfected with DNA encoding this di-leucine to di-alaninesubstituted GFP-botulinum A light chain. As can be seen, thesubstitution of alanine for leucine at positions 427 and 428 in thebotulinum type A light chain substantially changes the localizationcharacteristic of the light chain.

[0089] It is within the scope of this invention that a leucine-basedmotif, or any other persistence enhancing component and/or a biologicalactivity enhancing component present on a light chain, can be used toprotect the heavy chain as well. A random coil belt extends from thebotulinum type A translocation domain and encircles the light chain. Itis possible that this belt keeps the two subunits in proximity to eachother inside the cell while the light chain is localized to the cellmembrane. The structure of native botulinum toxin type A is shown inFIG. 6.

[0090] In addition, the data of the present invention shows that theleucine-based motif can be valuable in localizing the botulinum A toxinin close proximity to the SNAP-25 substrate within the cell. This canmean that the leucine-based motif is important not only for determiningthe half-life of the toxin but for determining the activity of the toxinas well. That is, the toxin will have a greater activity if it ismaintained in close proximity to the SNAP-25 substrate inside the cell.FIG. 5 shows the localization of SNAP-25 in horizontal confocal sectionsof differentiated PC12 cells (from Martinez-Arca et al (2000) J CellBiol 149:889-899). Similarity in the pattern of localization can be seenwhen comparing localization of botulinum type A light chain as seen inFIG. 1 to localization of SNAP-25 seen in FIG. 5.

[0091] The data of the present invention clearly shows that truncationof the light chain, thereby deleting the leucine-based motif, or aminoacid substitution within the leucine-based motif substantially changesmembrane localization of the botulinum type A light chain in nervecells. In both truncation and substitution a percentage of the alteredlight chain can localize to the cell membrane in a pattern unlike thatof the native type A light chain (see FIGS. 1, 2 and 4). This datasupports the presence of biological persistence enhancing componentsother than a leucine-based motif such as tyrosine motifs and amino acidderivatives. Use of these other biological persistence enhancingcomponents and/or a biological activity enhancing components in modifiedneurotoxins is also within the scope of the present invention.

[0092] Also within the scope of the present invention is more than onebiological persistence enhancing component used in combination in amodified neurotoxin to alter biological persistence of the neurotoxinthat is modified. The present invention also includes use of more thanone biological activity enhancing or biological activity reducingcomponents used in combination in a modified neurotoxin to alter thebiological activity of the neurotoxin that is modified.

[0093] Tyrosine-based motifs are within the scope of the presentinvention as biological persistence and/or a biological activityaltering components. Tyrosine-based motifs comprise the sequenceY-X-X-Hy where Y is tyrosine, X is any amino acid and Hy is ahydrophobic amino acid. Tyrosine-based motifs can act in a manner thatis similar to that of leucine-based motifs. In FIG. 3 some of tyrosinemotifs found in the type A toxin light chain are bracketed. In addition,a tyrosine-based motif is found within the leucine-based motif which isindicated by an asterisked bracket in FIG. 3.

[0094] Also within the scope of the present invention are modifiedneurotoxins which comprise one or more biological persistence alteringcomponents and/or a biological activity enhancing components which occurnaturally in both botulinum toxin types A and B.

[0095]FIG. 7 shows localization of GFP-botulinum type B neurotoxin lightchain in live, differentiated PC12 cells. Localization of the type Blight chain appears to be to an intracellular organelle. Similarlocalization pattern is seen for GFP-truncated botulinum type A shown inFIG. 2. Localization of a botulinum toxin, or botulinum toxin lightchain, within the cell is believed to be a key factor in determiningbiological persistence and/or biological activity of the toxin.Therefore, these data appear to indicate that there are biologicalpersistence altering component(s), and/or biological activity alteringcomponent(s), common to the type A and type B botulinum toxins. These,and other biological persistence altering components, and biologicalactivity altering components, are contemplated for use in accordancewith the present invention.

[0096]FIG. 8 shows a sequence alignment between type A and type B lightchains isolated from strains type A HallA (SEQ ID NO: 19) and type BDanish I (SEQ ID NO: 20) respectively. Light chains or heavy chainsisolated from other strains of botulinum toxin types A and B can also beused for sequence comparison. The shaded amino acids represent aminoacid identities, or matches, between the chains. Each of the shadedamino acids between amino acid position 10 and amino acid position 425of the FIG. 8 consensus sequence, alone or in combination with any othershaded amino acid or amino acids, represents a biological persistencealtering component that is within the scope of the present invention.For example, amino acids KAFK at positions 19 to 22, LNK at positions304 to 306, L at position 228 in combination with KL at positions 95 and96, FDKLYK at positions 346 to 351, YL-T at positions 78 to 81, YYD atpositions 73 to 75 in combination with YL at positions 78 and 79 incombination with T a position 81, F at position 297 in combination withI at position 300 in combination with KL at positions 95 and 96 can bebiological persistence altering components for use within the scope ofthis invention. In addition, conserved regions of charge,hydrophobicity, hydrophilicity and/or conserved secondary, tertiary, orquaternary structures that may be independent of conserved sequence arewithin the scope of the present invention.

[0097] Amino acid derivatives are also within the scope of the presentinvention as biological persistence enhancing components and/or asbiological activity enhancing components. Examples of amino acidderivatives that act to effect biological persistence and/or biologicalactivity are phosphorylated amino acids. These amino acids include, forexample, amino acids phosphorylated by tyrosine kinase, protein kinase Cor casein kinase II. Other amino acid derivatives within the scope ofthe present invention as biological persistence enhancing componentsand/or as biological activity enhancing components are myristylatedamino acids and N-glycosylated amino acids.

[0098] In one broad aspect of the present invention, a method isprovided for treating a condition using a modified neurotoxin. Theconditions can include, for example, skeletal muscle conditions, smoothmuscle conditions, pain and glandular conditions. The modifiedneurotoxin can also be used for cosmetics, for example, to treat browfurrows.

[0099] The neuromuscular disorders and conditions that can be treatedwith a modified neurotoxin include: for example, spasmodic dysphonia,laryngeal dystonia, oromandibular and lingual dystonia, cervicaldystonia, focal hand dystonia, blepharospasm, strabismus, hemifacialspasm, eyelid disorders, spasmodic torticolis, cerebral palsy, focalspasticity and other voice disorders, spasmodic colitis, neurogenicbladder, anismus, limb spasticity, tics, tremors, bruxism, anal fissure,achalasia, dysphagia and other muscle tone disorders and other disorderscharacterized by involuntary movements of muscle groups can be treatedusing the present methods of administration. Other examples ofconditions that can be treated using the present methods andcompositions are lacrimation, hyperhydrosis, excessive salivation andexcessive gastrointestinal secretions, as well as other secretorydisorders. In addition, the present invention can be used to treatdermatological conditions, for example, reduction of brow furrows,reduction of skin wrinkles. The present invention can also be used inthe treatment of sports injuries.

[0100] Borodic U.S. Pat. No. 5,053,005 discloses methods for treatingjuvenile spinal curvature, i.e. scoliosis, using botulinum type A. Thedisclosure of Borodic is incorporated in its entirety herein byreference. In one embodiment, using substantially similar methods asdisclosed by Borodic, a modified neurotoxin can be administered to amammal, preferably a human, to treat spinal curvature. In a preferredembodiment, a modified neurotoxin comprising botulinum type E fused witha leucine-based motif is administered. Even more preferably, a modifiedneurotoxin comprising botulinum type A-E with a leucine-based motiffused to the carboxyl terminal of its light chain is administered to themammal, preferably a human, to treat spinal curvature.

[0101] In addition, the modified neurotoxin can be administered to treatother neuromuscular disorders using well known techniques that arecommonly performed with botulinum type A. For example, the presentinvention can be used to treat pain, for example, headache pain, painfrom muscle spasms and various forms of inflammatory pain. For example,Aoki U.S. Pat. No. 5,721,215 and Aoki U.S. Pat. No. 6,113,915 disclosemethods of using botulinum toxin type A for treating pain. Thedisclosure of these two patents is incorporated in its entirety hereinby reference.

[0102] Autonomic nervous system disorders can also be treated with amodified neurotoxin. For example, glandular malfunctioning is anautonomic nervous system disorder. Glandular malfunctioning includesexcessive sweating and excessive salivation. Respiratory malfunctioningis another example of an autonomic nervous system disorder. Respiratorymalfunctioning includes chronic obstructive pulmonary disease andasthma. Sanders et al. disclose methods for treating the autonomicnervous system; for example, treating autonomic nervous system disorderssuch as excessive sweating, excessive salivation, asthma, etc., usingnaturally existing botulinum toxins. The disclosure of Sander et al. isincorporated in its entirety by reference herein. In one embodiment,substantially similar methods to that of Sanders et al. can be employed,but using a modified neurotoxin, to treat autonomic nervous systemdisorders such as the ones discussed above. For example, a modifiedneurotoxin can be locally applied to the nasal cavity of the mammal inan amount sufficient to degenerate cholinergic neurons of the autonomicnervous system that control the mucous secretion in the nasal cavity.

[0103] Pain that can be treated by a modified neurotoxin includes paincaused by muscle tension, or spasm, or pain that is not associated withmuscle spasm. For example, Binder in U.S. Pat. No. 5,714,468 disclosesthat headache caused by vascular disturbances, muscular tension,neuralgia and neuropathy can be treated with a naturally occurringbotulinum toxin, for example Botulinum type A. The disclosures of Binderare incorporated in its entirety herein by reference. In one embodiment,substantially similar methods to that of Binder can be employed, butusing a modified neurotoxin, to treat headache, especially the onescaused by vascular disturbances, muscular tension, neuralgia andneuropathy. Pain caused by muscle spasm can also be treated by anadministration of a modified neurotoxin. For example, a botulinum type Efused with a leucine-based motif, preferably at the carboxyl terminal ofthe botulinum type E light chain, can be administered intramuscularly atthe pain/spasm location to alleviate pain.

[0104] Furthermore, a modified neurotoxin can be administered to amammal to treat pain that is not associated with a muscular disorder,such as spasm. In one broad embodiment, methods of the present inventionto treat non-spasm related pain include central administration orperipheral administration of the modified neurotoxin.

[0105] For example, Foster et al. in U.S. Pat. No. 5,989,545 disclosesthat a botulinum toxin conjugated with a targeting moiety can beadministered centrally (intrathecally) to alleviate pain. Thedisclosures of Foster et al. are incorporated in its entirety byreference herein. In one embodiment, substantially similar methods tothat of Foster et al. can be employed, but using the modified neurotoxinaccording to this invention, to treat pain. The pain to be treated canbe an acute pain, or preferably, chronic pain.

[0106] An acute or chronic pain that is not associated with a musclespasm can also be alleviated with a local, peripheral administration ofthe modified neurotoxin to an actual or a perceived pain location on themammal. In one embodiment, the modified neurotoxin is administeredsubcutaneously at or near the location of pain, for example, at or neara cut. In another embodiment, the modified neurotoxin is administeredintramuscularly at or near the location of pain, for example, at or neara bruise location on the mammal. In another embodiment, the modifiedneurotoxin is injected directly into a joint of a mammal, for treatingor alleviating pain caused by arthritic conditions. Also, frequentrepeated injection or infusion of the modified neurotoxin to aperipheral pain location is within the scope of the present invention.However, given the long lasting therapeutic effects of the presentinvention, frequent injection or infusion of the neurotoxin can not benecessary. For example, practice of the present invention can provide ananalgesic effect, per injection, for 2 months or longer, for example 27months, in humans.

[0107] Without wishing to limit the invention to any mechanism or theoryof operation, it is believed that when the modified neurotoxin isadministered locally to a peripheral location, it inhibits the releaseof Neuro-substances, for example substance P, from the peripheralprimary sensory terminal by inhibiting SNARE-dependent exocytosis. Sincethe release of substance P by the peripheral primary sensory terminalcan cause or at least amplify pain transmission process, inhibition ofits release at the peripheral primary sensory terminal will dampen thetransmission of pain signals from reaching the brain.

[0108] In addition to having pharmacologic actions at the peripherallocation, the modified neurotoxin of the present invention can also haveinhibitory effects in the central nervous system, upon directintrathecal administration, as set forth in U.S. Pat. No. 6,113,915, orupon peripheral administration, where presumably the modified toxin actsthrough retrograde transport via a primary sensory afferent. Thishypothesis of retrograde axonal transport is supported by published datawhich shows that botulinum type A can be retrograde transported to thedorsal horn when the neurotoxin is injected peripherally. Thus, work byWeigand et al, Nauny-Schmiedeberg's Arch. Pharmacol. 1976; 292, 161-165,and Habermann, Nauny-Schmiedeberg's Arch. Pharmacol. 1974; 281, 47-56,showed that botulinum toxin is able to ascend to the spinal area byretrograde transport. As such, a modified neurotoxin, for examplebotulinum type A with one or more amino acids mutated from theleucine-based motif, injected at a peripheral location, for exampleintramuscularly, can be expected to be retrograde transported from theperipheral primary sensory terminal to a central region.

[0109] The amount of the modified neurotoxin administered can varywidely according to the particular disorder being treated, its severityand other various patient variables including size, weight, age, andresponsiveness to therapy. Generally, the dose of modified neurotoxin tobe administered will vary with the age, presenting condition and weightof the mammal, preferably a human, to be treated. The potency of themodified neurotoxin will also be considered.

[0110] Assuming a potency (for a botulinum toxin type A) which issubstantially equivalent to LD₅₀=2,730 U in a human patient and anaverage person is 75 kg, a lethal dose (for a botulinum toxin type A)would be about 36 U/kg of a modified neurotoxin. Therefore, when amodified neurotoxin with such an LD₅₀ is administered, it would beappropriate to administer less than 36 U/kg of the modified neurotoxininto human subjects. Preferably, about 0.01 U/kg to 30 U/kg of themodified neurotoxin is administered. More preferably, about 1 U/kg toabout 15 U/kg of the modified neurotoxin is administered. Even morepreferably, about 5 U/kg to about 10 U/kg modified neurotoxin isadministered. Generally, the modified neurotoxin will be administered asa composition at a dosage that is proportionally equivalent to about 2.5cc/100 U. Those of ordinary skill in the art will know, or can readilyascertain, how to adjust these dosages for neurotoxin of greater orlesser potency. It is known that botulinum toxin type B can beadministered at a level about fifty times higher that that used for abotulinum toxin type A for similar therapeutic effect. Thus, the unitsamounts set forth above can be multiplied by a factor of about fifty fora botulinum toxin type B.

[0111] Although examples of routes of administration and dosages areprovided, the appropriate route of administration and dosage aregenerally determined on a case by case basis by the attending physician.Such determinations are routine to one of ordinary skill in the art (seefor example, Harrison's Principles of Internal Medicine (1998), editedby Anthony Fauci et al., 14^(th) edition, published by McGraw Hill). Forexample, the route and dosage for administration of a modifiedneurotoxin according to the present disclosed invention can be selectedbased upon criteria such as the solubility characteristics of themodified neurotoxin chosen as well as the types of disorder beingtreated.

[0112] The modified neurotoxin can be produced by chemically linking theleucine-based motif to a neurotoxin using conventional chemical methodswell known in the art. For example, botulinum type E can be obtained byestablishing and growing cultures of Clostridium botulinum in afermenter, and then harvesting and purifying the fermented mixture inaccordance with known procedures.

[0113] The modified neurotoxin can also be produced by recombinanttechniques. Recombinant techniques are preferable for producing aneurotoxin having amino acid sequence regions from different Clostridialspecies or having modified amino acid sequence regions. Also, therecombinant technique is preferable in producing botulinum type A withthe leucine-based motif being modified by deletion. The techniqueincludes steps of obtaining genetic materials from natural sources, orsynthetic sources, which have codes for a cellular binding moiety, anamino acid sequence effective to translocate the neurotoxin or a partthereof, and an amino acid sequence having therapeutic activity whenreleased into a cytoplasm of a target cell, preferably a neuron. In apreferred embodiment, the genetic materials have codes for thebiological persistence enhancing component, preferably the leucine-basedmotif, the H_(C), the H_(N) and the light chain of the Clostridialneurotoxins and fragments thereof. The genetic constructs areincorporated into host cells for amplification by first fusing thegenetic constructs with a cloning vectors, such as phages or plasmids.

[0114] Then the cloning vectors are inserted into a host, for example,Clostridium sp., E. coli or other prokaryotes, yeast, insect cell lineor mammalian cell lines. Following the expressions of the recombinantgenes in host cells, the resultant proteins can be isolated usingconventional techniques.

[0115] There are many advantages to producing these modified neurotoxinsrecombinantly. For example, to form a modified neurotoxin, a modifyingfragment, or component must be attached or inserted into a neurotoxin.The production of neurotoxin from anaerobic Clostridium cultures is acumbersome and time-consuming process including a multi-steppurification protocol involving several protein precipitation steps andeither prolonged and repeated crystallization of the toxin or severalstages of column chromatography. Significantly, the high toxicity of theproduct dictates that the procedure must be performed under strictcontainment (BL-3). During the fermentation process, the foldedsingle-chain neurotoxins are activated by endogenous Clostridialproteases through a process termed nicking to create a dichain.Sometimes, the process of nicking involves the removal of approximately10 amino acid residues from the single-chain to create the dichain formin which the two chains remain covalently linked through the intrachaindisulfide bond.

[0116] The nicked neurotoxin is much more active than the unnicked form.The amount and precise location of nicking varies with the serotypes ofthe bacteria producing the toxin. The differences in single-chainneurotoxin activation and, hence, the yield of nicked toxin, are due tovariations in the serotype and amounts of proteolytic activity producedby a given strain. For example, greater than 99% of Clostridialbotulinum serotype A single-chain neurotoxin is activated by the Hall AClostridial botulinum strain, whereas serotype B and E strains producetoxins with lower amounts of activation (0 to 75% depending upon thefermentation time). Thus, the high toxicity of the mature neurotoxinplays a major part in the commercial manufacture of neurotoxins astherapeutic agents.

[0117] The degree of activation of engineered Clostridial toxins is,therefore, an important consideration for manufacture of thesematerials. It would be a major advantage if neurotoxins such asbotulinum toxin and tetanus toxin could be expressed, recombinantly, inhigh yield in rapidly-growing bacteria (such as heterologous E. colicells) as relatively non-toxic single-chains (or single chains havingreduced toxic activity) which are safe, easy to isolate and simple toconvert to the fully-active form.

[0118] With safety being a prime concern, previous work has concentratedon the expression in E. coli and purification of individual H and lightchains of tetanus and botulinum toxins; these isolated chains are, bythemselves, non-toxic; see Li et al., Biochemistry 33:7014-7020 (1994);Zhou et al., Biochemistry 34:15175-15181 (1995), hereby incorporated byreference herein.

[0119] Following the separate production of these peptide chains andunder strictly controlled conditions the H and light chains can becombined by oxidative disulphide linkage to form the neuroparalyticdi-chains.

EXAMPLES

[0120] The following non-limiting examples provide those of ordinaryskill in the art with specific preferred methods to treat non-spasmrelated pain within the scope of the present invention and are notintended to limit the scope of the invention.

Example 1 Treatment of Pain Associated with Muscle Disorder

[0121] An unfortunate 36 year old woman has a 15 year history oftemporomandibular joint disease and chronic pain along the masseter andtemporalis muscles. Fifteen years prior to evaluation she notedincreased immobility of the jaw associated with pain and jaw opening andclosing and tenderness along each side of her face. The lef t side isoriginally thought to be worse than the right. She is diagnosed ashaving temporomandibular joint (TMJ) dysfunction with subluxation of thejoint and is treated with surgical orthoplasty meniscusectomy andcondyle resection.

[0122] She continues to have difficulty with opening and closing her jawafter the surgical procedures and for this reason, several years later,a surgical procedure to replace prosthetic joints on both sides isperformed. After the surgical procedure progressive spasms and deviationof the jaw ensues. Further surgical revision is performed subsequent tothe original operation to correct prosthetic joint loosening. The jawcontinues to exhibit considerable pain and immobility after thesesurgical procedures. The TMJ remained tender as well as the muscleitself. There are tender points over the temporomandibular joint as wellas increased tone in the entire muscle. She is diagnosed as havingpost-surgical myofascial pain syndrome and is injected with the modifiedneurotoxin into the masseter and temporalis muscles; the modifiedneurotoxin is botulinum type E comprising a leucine-based motif. Theparticular dose as well as the frequency of administrations depends upona variety of factors within the skill of the treating physician.

[0123] Several days after the injections she noted substantialimprovement in her pain and reports that her jaw feels looser. Thisgradually improves over a 2 to 3 week period in which she notesincreased ability to open the jaw and diminishing pain. The patientstates that the pain is better than at any time in the last 4 years. Theimproved condition persists for up to 27 months after the originalinjection of the modified neurotoxin.

Example 2 Treatment of Pain Subsequent to Spinal Cord Injury

[0124] A patient, age 39, experiencing pain subsequent to spinal cordinjury is treated by intrathecal administration, for example, by spinaltap or by catherization (for infusion) to the spinal cord, with themodified neurotoxin; the modified neurotoxin is botulinum type Ecomprising a leucine-based motif. The particular toxin dose and site ofinjection, as well as the frequency of toxin administrations, dependupon a variety of factors within the skill of the treating physician, aspreviously set forth. Within about 1 to about 7 days after the modifiedneurotoxin administration, the patient's pain is substantially reduced.The pain alleviation persists for up to 27 months.

Example 3 Peripheral Administration of a Modified Neurotoxin to Treat“Shoulder-Hand Syndrome”

[0125] Pain in the shoulder, arm, and hand can develop, with musculardystrophy, osteoporosis and fixation of joints. While most common aftercoronary insufficiency, this syndrome can occur with cervicalosteoarthritis or localized shoulder disease, or after any prolongedillness that requires the patient to remain in bed.

[0126] A 46 year old woman presents a shoulder-hand syndrome type pain.The pain is particularly localized at the deltoid region. The patient istreated by a bolus injection of a modified neurotoxin subcutaneously tothe shoulder; preferably the modified neurotoxin is botulinum type Ecomprising a leucine-based motif. The modified neurotoxin can also be,for example, modified botulinum type A, B, C1, C2, D, E, F or G whichcomprise a leucine-based motif. The particular dose as well as thefrequency of administrations depends upon a variety of factors withinthe skill of the treating physician, as previously set forth. Within 1-7days after modified neurotoxin administration the patient's pain issubstantially alleviated. The duration of the pain alleviation is fromabout 7 to about 27 months.

Example 4 Peripheral Administration of a Modified Neurotoxin to TreatPostherapeutic Neuralgia

[0127] Postherapeutic neuralgia is one of the most intractable ofchronic pain problems. Patients suffering this excruciatingly painfulprocess often are elderly, have debilitating disease, and are notsuitable for major interventional procedures. The diagnosis is readilymade by the appearance of the healed lesions of herpes and by thepatient's history. The pain is intense and emotionally distressing.Postherapeutic neuralgia can occur anywhere, but is most often in thethorax.

[0128] A 76 year old man presents a postherapeutic type pain. The painis localized to the abdomen region. The patient is treated by a bolusinjection of a modified neurotoxin intradermally to the abdomen; themodified neurotoxin is, for example, botulinum type A, B, C1, C2, D, E,F and/or G. The modified neurotoxin comprises a leucine-based motifand/or additional tyrosine-based motifs. The particular dose as well asthe frequency of administration depends upon a variety of factors withinthe skill of the treating physician, as previously set forth. Within 1-7days after modified neurotoxin administration the patient's pain issubstantially alleviated. The duration of the pain alleviation is fromabout 7 to about 27 months.

Example 5 Peripheral Administration of a Modified Neurotoxin to TreatNasopharyngeal Tumor Pain

[0129] These tumors, most often squamous cell carcinomas, are usually inthe fossa of Rosenmuller and can invade the base of the skull. Pain inthe face is common. It is constant, dull-aching in nature.

[0130] A 35 year old man presents a nasopharyngeal tumor type pain. Painis found at the lower left cheek. The patient is treated by a bolusinjection of a modified neurotoxin intramuscularly to the cheek,preferably the modified neurotoxin is botulinum type A, B, C1, C2, D, E,F or G comprising additional biological persistence enhancing amino acidderivatives, for example, tyrosine phosphorylations. The particular doseas well as the frequency of administrations depends upon a variety offactors within the skill of the treating physician. Within 1-7 daysafter modified neurotoxin administration the patient's pain issubstantially alleviated. The duration of the pain alleviation is fromabout 7 to about 27 months.

Example 6 Peripheral Administration of a Modified Neurotoxin to TreatInflammatory Pain

[0131] A patient, age 45, presents an inflammatory pain in the chestregion. The patient is treated by a bolus injection of a modifiedneurotoxin intramuscularly to the chest, preferably the modifiedneurotoxin is botulinum type A, B, C1, C2, D, E, F or G comprisingadditional tyrosine-based motifs. The particular dose as well as thefrequency of administrations depends upon a variety of factors withinthe skill of the treating physician, as previously set forth. Within 1-7days after modified neurotoxin administration the patient's pain issubstantially alleviated. The duration of the pain alleviation is fromabout 7 to about 27 months.

Example 7 Treatment of Excessive Sweating

[0132] A male, age 65, with excessive unilateral sweating is treated byadministering a modified neurotoxin. The dose and frequency ofadministration depends upon degree of desired effect. Preferably, themodified neurotoxin is botulinum type A, B, C1, C2, D, E, F and/or G.The modified neurotoxins comprise a leucine-based motif. Theadministration is to the gland nerve plexus, ganglion, spinal cord orcentral nervous system. The specific site of administration is to bedetermined by the physician's knowledge of the anatomy and physiology ofthe target glands and secretory cells. In addition, the appropriatespinal cord level or brain area can be injected with the toxin. Thecessation of excessive sweating after the modified neurotoxin treatmentis up to 27 months.

Example 8 Post Surgical Treatments

[0133] A female, age 22, presents a torn shoulder tendon and undergoesorthopedic surgery to repair the tendon. After the surgery, the patientis administered intramuscularly with a modified neurotoxin to theshoulder. The modified neurotoxin can botulinum type A, B, C, D, E, F,and/or G wherein one or more amino acids of a biological persistenceenhancing component are deleted from the toxin. For example, one or moreleucine residues can be deleted from and/or mutated from theleucine-based motif in botulinum toxin serotype A. Alternatively, one ormore amino acids of the leucine-based motif can be substituted for otheramino acids. For example, the two leucines in the leucine-based motifcan be substituted for alanines. The particular dose as well as thefrequency of administrations depends upon a variety of factors withinthe skill of the treating physician. The specific site of administrationis to be determined by the physician's knowledge of the anatomy andphysiology of the muscles. The administered modified neurotoxin reducesmovement of the arm to facilitate the recovery from the surgery. Theeffect of the modified neurotoxin is for about five weeks or less.

Example 9 Cloning, Expression and Purification of the BotulinumNeurotoxin Light Chain Gene

[0134] This example describes methods to clone and express a DNAnucleotide sequence encoding a botulinum toxin light chain and purifythe resulting protein product. A DNA sequence encoding the botulinumtoxin light chain can be amplified by PCR protocols which employsynthetic oligonucleotides having sequences corresponding to the 5′ and3′ end regions of the light chain gene. Design of the primers can allowfor the introduction of restriction sites, for example, Stu I and EcoR Irestriction sites into the 5′ and 3′ ends of the botulinum toxin lightchain gene PCR product. These restriction sites can be subsequently usedto facilitate unidirectional subcloning of the amplification products.Additionally, these primers can introduce a stop codon at the C-terminusof the light chain coding sequence. Chromosomal DNA from C. botulinum,for example, strain HallA, can serve as a template in the amplificationreaction.

[0135] The PCR amplification can be performed in a 0.1 mL volumecontaining 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM ofeach deoxynucleotide triphosphate (dNTP), 50 pmol of each primer, 200 ngof genomic DNA and 2.5 units of Taq DNA polymerase. The reaction mixturecan be subjected to 35 cycles of denaturation (1 minute at 94° C.),annealing (2 minutes at 55° C.) and polymerization (2 minutes at 72°C.). Finally, the reaction can be extended for an additional minutes at72° C.

[0136] The PCR amplification product can be digested with for example,Stu I and EcoR I, to release the light chain encoding, cloned, PCR DNAfragment. This fragment can then be purified by, for example, agarosegel electrophoresis, and ligated into, for example, a Sma I and EcoR Idigested pBluescript II SK phagemid. Bacterial transformants, forexample, E coli, harboring this recombinant phagemid can be identifiedby standard procedures, such as blue/white screening. Clones comprisingthe light chain encoding DNA can be identified by DNA sequence analysisperformed by standard methods. The cloned sequences can be confirmed bycomparing the cloned sequences to published sequences for botulinumlight chains, for example, Binz, et al., in J. Biol. Chem. 265, 9153(1990), Thompson et al., in Eur. J. Biochem. 189, 73 (1990) and Minton,Clostridial Neurotoxins, The Molecular Pathogenesis of Tetanus andBotulism p. 161-191, Edited by C. Motecucco (1995).

[0137] The light chain can be subcloned into an expression vector, forexample, pMal-P2. pMal-P2 harbors the malE gene encoding MBP (maltosebinding protein) which is controlled by a strongly inducible promoter,P_(tac).

[0138] To verify expression of the botulinum toxin light chain, a wellisolated bacterial colony harboring the light chain gene containingpMal-P2 can be used to inoculate L-broth containing 0.1 mg/ml ampicillinand 2% (w/v) glucose, and grown overnight with shaking at 30° C. Theovernight cultures can be diluted 1:10 into fresh L-broth containing 0.1mg/ml of ampicillin and incubated for 2 hours. Fusion protein expressioncan be induced by addition of IPTG to a final concentration of 0.1 mM.After an additional 4 hour incubation at 30° C., bacteria can becollected by centrifugation at 6,000× g for 10 minutes.

[0139] A small-scale SDS-PAGE analysis can confirm the presence of a 90kDa protein band in samples derived from IPTG-induced bacteria. This MWwould be consistent with the predicted size of a fusion protein havingMBP (˜40 kDa) and botulinum toxin light chain (˜50 kDa) components.

[0140] The presence of the desired fusion proteins in IPTG-inducedbacterial extracts can be confirmed by western blotting using thepolyclonal anti-L chain probe described by Cenci di Bello et al., inEur. J. Biochem. 219, 161 (1993). Reactive bands on PVDF membranes(Pharmacia; Milton Keynes, UK) can be visualized using an anti-rabbitimmunoglobulin conjugated to horseradish peroxidase (BioRad; HemelHempstead, UK) and the ECL detection system (Amersham, UK). Westernblotting results typically confirm the presence of the dominant fusionprotein together with several faint bands corresponding to proteins oflower MW than the fully sized fusion protein. This observation suggeststhat limited degradation of the fusion protein occurred in the bacteriaor during the isolation procedure.

[0141] To produce the subcloned light chain, pellets from 1 litercultures of bacteria expressing the wild-type Botulinum neurotoxin lightchain proteins can be resuspended in column buffer [10 mM Tris-HCl (pH8.0), 200 mM NaCl, 1 mM EGTA and 1 mM DTT] containing 1 mMphenylmethanesulfonyl fluoride (PMSF) and 10 mM benzamidine, and lysedby sonication. The lysates can be cleared by centrifugation at 15,000× gfor 15 minutes at 4° C. Supernatants can be applied to an amyloseaffinity column [2×10 cm, 30 ml resin] (New England BioLabs; Hitchin,UK). Unbound proteins can be washed from the resin with column bufferuntil the eluate is free of protein as judged by a stable absorbancereading at 280 nm. The bound MBP-L chain fusion protein can besubsequently eluted with column buffer containing 10 mM maltose.Fractions containing the fusion protein can be pooled and dialyzedagainst 20 mM Tris-HCl (pH 8.0) supplemented with 150 mM NaCl, 2 mM,CaCl2 and 1 mM DTT for 72 hours at 4° C.

[0142] The MBP-L chain fusion proteins can be purified after releasefrom the host bacteria. Release from the bacteria can be accomplished byenzymatically degrading or mechanically disrupting the bacterial cellmembrane. Amylose affinity chromatography can be used for purification.Recombinant wild-type or mutant light chains can be separated from thesugar binding domains of the fusion proteins by site-specific cleavagewith Factor Xa. This cleavage procedure typically yields free MBP, freelight chains and a small amount of uncleaved fusion protein. While theresulting light chains present in such mixtures can be shown to possessthe desired activities, an additional purification step can be employed.For example, the mixture of cleavage products can be applied to a secondamylose affinity column which binds both the MBP and uncleaved fusionprotein. Free light chains can be isolated in the flow through fraction.

Example 10 Reconstitution of Native Light Chain. Recombinant Wild-TypeLight Chain with Purified Heavy Chain

[0143] Native heavy and light chains can be dissociated from a BoNT with2 M urea, reduced with 100 mM DTT and then purified according toestablished chromatographic procedures. For example, Kozaki et al.(1981, Japan J. Med. Sci. Biol. 34, 61) and Maisey et al. (1988, Eur. J.Biochem. 177, 683). A purified heavy chain can be combined with anequimolar amount of either native light chain or a recombinant lightchain. Reconstitution can be carried out by dialyzing the samplesagainst a buffer consisting of 25 mM Tris (pH 8.0), 50 μM zinc acetateand 150 mM NaCl over 4 days at 4° C. Following dialysis, the associationof the recombinant light chain and native heavy chain to form disulfidelinked 150 kDa dichains is monitored by SDS-PAGE and/or quantified bydensitometric scanning.

Example 11 Production of a Modified Neurotoxin with an EnhancedBiological Persistence

[0144] A modified neurotoxin can be produced by employing recombinanttechniques in conjunction with conventional chemical techniques.

[0145] A neurotoxin chain, for example a botulinum light chain that isto be fused with a biological persistence enhancing component to form amodified neurotoxin can be produced recombinantly and purified asdescribed in example 9.

[0146] The recombinant neurotoxin chain derived from the recombinanttechniques can be covalently fused with (or coupled to) a biologicalpersistence enhancing component, for example a leucine-based motif, atyrosine-based motif and/or an amino acid derivative. Peptide sequencescomprising biological persistence enhancing components can besynthesized by standard t-Boc/Fmoc technologies in solution or solidphase as is known to those skilled in the art. Similar synthesistechniques are also covered by the scope of this invention, for example,methodologies employed in Milton et al. (1992, Biochemistry 31,8799-8809) and Swain et al. (1993, Peptide Research 6, 147-154). One ormore synthesized biological persistence enhancing components can befused to the light chain of botulinum type A, B, C1, C2, D, E, F or Gat, for example, the carboxyl terminal end of the toxin. The fusion ofthe biological persistence enhancing components is achieved by chemicalcoupling using reagents and techniques known to those skilled in theart, for example PDPH/EDAC and Traut's reagent chemistry.

[0147] Alternatively, a modified neurotoxin can be producedrecombinantly without the step of fusing the biological persistenceenhancing component to a recombinant botulinum toxin chain. For example,a recombinant neurotoxin chain, for example, a botulinum light chain,derived from the recombinant techniques of example 9 can be producedwith a biological persistence enhancing component, for example aleucine-based motif, a tyrosine-based motif and/or an amino acidderivative. For example, one or more DNA sequences encoding biologicalpersistence enhancing components can be added to the DNA sequenceencoding the light chain of botulinum type A, B, C1, C2, D, E, F or G.This addition can be done by any number of methods used for sitedirected mutagenesis which are familiar to those skilled in the art.

[0148] The recombinant modified light chain containing the fused oradded biological persistence enhancing component can be reconstitutedwith a heavy chain of a neurotoxin by the method described in example 10thereby producing a complete modified neurotoxin.

[0149] The modified neurotoxins produced according to this example havean enhanced biological persistence. Preferably, the biologicalpersistence is enhanced by about 20% to about 300% relative to anidentical neurotoxin without the additional biological persistenceenhancing component(s).

Example 12 Production of a Modified Neurotoxin with a Reduced BiologicalPersistence

[0150] A modified neurotoxin with a reduced biological persistence canbe produced by employing recombinant techniques. For example, abotulinum light chain derived from the recombinant techniques of example9 can be produced without a biological persistence enhancing component.For example, one or more leucine-based motifs, tyrosine-based motifsand/or amino acid derivatives can be mutated. For example, one or moreDNA sequences encoding biological persistence enhancing components canbe removed from the DNA sequence encoding the light chain of botulinumtype A, B, C1, C2, D, E, F or G. For example, the DNA sequence encodingthe leucine based motif can be removed from the DNA sequence encodingbotulinum type A light chain. Removal of the DNA sequences can be doneby any number of methods familiar to those skilled in the art.

[0151] The recombinant modified light chain with the deleted biologicalpersistence enhancing component can be reconstituted with a heavy chainof a neurotoxin by the method described in example 10 thereby producinga complete modified neurotoxin.

[0152] The modified neurotoxin produced according to this example has areduced biological persistence. Preferably, the biological persistenceis reduced by about 20% to about 300% relative to an identicalneurotoxin, for example botulinum type A, with the leucine-based motif.

[0153] Although the present invention has been described in detail withregard to certain preferred methods, other embodiments, versions, andmodifications within the scope of the present invention are possible.For example, a wide variety of modified neurotoxins can be effectivelyused in the methods of the present invention in place of Clostridialneurotoxins. Also, the corresponding genetic codes, i.e. DNA sequence,to the modified neurotoxins are also considered to be part of thisinvention. Additionally, the present invention includes peripheraladministration methods wherein two or more modified neurotoxins, forexample botulinum type E with a fused leucine-based motif and botulinumtype B comprising a leucine-based motif, are administered concurrentlyor consecutively. While this invention has been described with respectto various specific examples and embodiments, it is to be understoodthat the invention is not limited thereto and that it can be variouslypracticed with the scope of the following claims.

Example 13 Production of a Modified Neurotoxin with a Reduced BiologicalPersistence

[0154] Localization to the cellular membrane is likely a key factor indetermining the biological persistence of botulinum toxins. This isbecause localization to a cell membrane can protect the localizedprotein from inter-cellular protein degrading complexes.

[0155] It is well known and generally accepted that the biologicalpersistence of botulinum type B neurotoxin is shorter than thebiological persistence of botulinum type A neurotoxin. In this work, itwas demonstrated that when the botulinum toxin type A light chain istruncated, which comprises removing the leucine-based motif, the lightchain substantially loses its ability to localize to the cellularmembrane in its characteristic pattern. In fact, truncated type A lightchain localizes to the cellular membrane in a pattern similar to that ofbotulinum toxin type B light chain.

[0156] Therefore, it can be hypothesized that truncated botulinum type Ahas a reduced biological persistence and/or a reduced biologicalactivity similar to that of botulinum toxin type B.

Example 14 Production of a Modified Neurotoxin with an AlteredBiological Persistence

[0157] Localization to the cellular membrane is likely a key factor indetermining the biological persistence of botulinum toxins. This isbecause localization to a cell membrane can protect the localizedprotein from inter-cellular protein degrading complexes.

[0158] In this work, it was demonstrated that when the botulinum toxintype A light chain is mutated, changing the two leucines at positions427 and 428 to alanines (FIG. 3), the light chain substantially losesits ability to localize to the cellular membrane in its characteristicpattern.

[0159] From this data it can be concluded that the mutated botulinumtype A has an altered biological persistence.

Example 15 In vitro Cleavage of SNAP 25 by Truncated LC/A

[0160] As illustrated by FIG. 9, an in vitro ELISA assay was carried outby the inventors demonstrating that a truncated LC/A in vitro cleavesSNAP-25 substrate less efficiently than does non-truncated LC/A. Thedata displayed is not a measure of inhibition of exocytosis but ameasure of the in vitro formation of SNAP-25 cleavage. The assay wascarried out as follows:

[0161] Materials:

[0162] BirA-SNAP25₁₂₈₋₂₀₆—this is a recombinant substrate for LC/A,consisting of a BirA signal sequence fused to the N-terminus of residues128-206 of SNAP25. This fusion construct was produced in E. coli and theBirA signal sequence was biotinylated by the E. coli. Microtiter plateswere coated with streptavidin. The toxin used was BoNT/A complex or LC/Aconstructs. The primary antibody was anti-SNAP25₁₉₇ antibody. Thisantibody recognizes the C-terminus of SNAP25 following cleavage by TypeA toxin (BirA-SNAP25₁₂₈₋₁₉₇). The secondary antibody was goat,anti-rabbit IgG conjugated to horseradish peroxidase. The ImmunoPure TMBsubstrate was from Pierce, a colorimetric substrate for horseradishperoxidase. The antibody that recognizes the cleaved product SNAP25₁₉₇is specific for that cleaved product and does not recognize the fulllength uncleaved substrate SNAP25₂₀₆.

[0163] Method:

[0164] BirA-SNAP25₁₂₈₋₂₀₆ was bound to streptavidin on a microtiterplate. To the plates were added serial dilutions of BoNT/A 900 kDacomplex, His6-S-nativeLC/A, or His6-S-truncLC/A-His6. All toxin sampleswere pre-incubated with DTT (this is not required for the LC/Aconstructs, but they were treated the same as the BoNT/A complex). Thetoxin samples were incubated with the substrate for 90 minutes at 37° C.The toxin was removed and the bound substrate was incubated withanti-SNAP25₁₉₇ antibody. Unbound antibody was washed away and the plateswere then incubated with the secondary antibody (anti-rabbit IgGconjugated to horseradish peroxidase). Unbound antibody was again washedaway and a calorimetric assay for horseradish peroxidase was performed.The assay was quantified by reading the absorbance at 450 nm.

[0165] In other work by the inventors disclosed herein the light chainconstructs that were expressed in the PC-12 cells were expresseddirectly in the PC-12 cells and do not contain any tags. The light chainconstructs that have been expressed from E. coli for these in vitroassays contain affinity tags for purification purposes (these tags arenot present on the proteins expressed in the PC-12 cells, as disclosedherein). The LC/A expressed in PC12 was the fusion protein GFP-LC/A.Between the GFP and the LC/A there is a set of Gly to separate bothproteins.

[0166] An explanation of the various constructs follows:

[0167] Complex (red in the graph) this is BoNT/A 900 kDa complexisolated from C. botulinum

[0168] Truncated LC/A—construct lacking 8 amino acids at the N-terminusand 22 amino acids at the C-terminus. However, this construct doescontain a 6-histidine and an S-tag at the N-terminus as well as a6-histidine tag at the C-terminus.

[0169] Dialyzed Truncated LC/A—same as Truncated LC/A, but imidazoleresulting from the purification has been removed.

[0170] Full LC/A (Dark green in graph)—native LC/A construct(full-length), but containing the N-terminal 6-histidine and S-tag. Doesnot have the C-terminal 6-histidine.

[0171] Dialyzed Full LC/A (Light green in graph)—Same as Full LC/A, butimidazole resulting from the purification has been removed.

[0172] To graphically depict these differences, FIG. 10 shows the veryN-terminus and the very C-terminus of these constructs (the middleportion of the LC/A proteins is not shown). What is referred to asWildtype corresponds to the native LC/A that the inventors had expresseddirectly in the PC-12 cells (this is construct that the inventorsdemonstrated activity with via Western blot analysis of the cleavedSNAP25 product). Truncated LC/A is the truncated light chain containingthe His and S-tags. N-His-LC/A is what was referred to as Full LC/A inFIG. 9.

1 20 1 7 PRT Artificial Sequence MISC_FEATURE (1)..(5) Description ofArtificial Sequence fragment having properties substantially similar tothat of leucine based sequence x may be any amino acid or derivativesthereof 1 Xaa Asp Xaa Xaa Xaa Leu Leu 1 5 2 7 PRT Artificial SequenceMISC_FEATURE (1)..(5) Description of Artificial Sequence fragment havingproperties substantially similar to leucine based motif x may be anyamino acid or derivatives thereof 2 Xaa Glu Xaa Xaa Xaa Leu Leu 1 5 3 7PRT Artificial Sequence MISC_FEATURE (1)..(5) Description of ArtificialSequence fragment having properties substantially similar to that ofleucine based motif 3 Xaa Asp Xaa Xaa Xaa Leu Ile 1 5 4 7 PRT ArtificialSequence MISC_FEATURE (1)..(5) Description of Artificial Sequencefragment having properties substantially similar to that of leucinebased motif 4 Xaa Asp Xaa Xaa Xaa Leu Met 1 5 5 7 PRT ArtificialSequence MISC_FEATURE (1)..(5) Description of Artificial Sequencefragment having properties substantially similar to leucine based motif5 Xaa Glu Xaa Xaa Xaa Leu Ile 1 5 6 7 PRT Unknown MISC_FEATURE (1)..(5)Description of Unknown Organism This fragment may have come from a ratsource. 6 Xaa Glu Xaa Xaa Xaa Leu Met 1 5 7 7 PRT Unknown Description ofUnknown Organism This fragment may have come from a rat source. 7 PheGlu Phe Tyr Lys Leu Leu 1 5 8 7 PRT rat 8 Glu Glu Lys Arg Ala Ile Leu 15 9 7 PRT rat 9 Glu Glu Lys Met Ala Ile Leu 1 5 10 7 PRT rat 10 Ser GluArg Asp Val Leu Leu 1 5 11 7 PRT rat 11 Val Asp Thr Gln Val Leu Leu 1 512 7 PRT mouse 12 Ala Glu Val Gln Ala Leu Leu 1 5 13 7 PRT frog 13 SerAsp Lys Gln Asn Leu Leu 1 5 14 7 PRT chicken 14 Ser Asp Arg Gln Asn LeuIle 1 5 15 7 PRT sheep 15 Ala Asp Thr Gln Val Leu Met 1 5 16 7 PRT Homosapiens 16 Ser Asp Lys Gln Thr Leu Leu 1 5 17 7 PRT Homo sapiens 17 SerGln Ile Lys Arg Leu Leu 1 5 18 7 PRT Homo sapiens 18 Ala Asp Thr Gln AlaLeu Leu 1 5 19 437 PRT Clostridium botulinum 19 Pro Phe Val Asn Lys GlnPhe Asn Tyr Lys Asp Pro Val Asn Gly Val 1 5 10 15 Asp Ile Ala Tyr IleLys Ile Pro Asn Val Gly Gln Met Gln Pro Val 20 25 30 Lys Ala Phe Lys IleHis Asn Lys Ile Trp Val Ile Pro Glu Arg Asp 35 40 45 Thr Phe Thr Asn ProGlu Glu Gly Asp Leu Asn Pro Pro Pro Glu Ala 50 55 60 Lys Gln Val Pro ValSer Tyr Tyr Asp Ser Thr Tyr Leu Ser Thr Asp 65 70 75 80 Asn Glu Lys AspAsn Tyr Leu Lys Gly Val Thr Lys Leu Phe Glu Arg 85 90 95 Ile Tyr Ser ThrAsp Leu Gly Arg Met Leu Leu Thr Ser Ile Val Arg 100 105 110 Gly Ile ProPhe Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu Lys Val 115 120 125 Ile AspThr Asn Cys Ile Asn Val Ile Gln Pro Asp Gly Ser Tyr Arg 130 135 140 SerGlu Glu Leu Asn Leu Val Ile Ile Gly Pro Ser Ala Asp Ile Ile 145 150 155160 Gln Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn Leu Thr Arg 165170 175 Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro Asp Phe Thr180 185 190 Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro Leu LeuGly 195 200 205 Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr Leu Ala HisGlu Leu 210 215 220 Ile His Ala Gly His Arg Leu Tyr Gly Ile Ala Ile AsnPro Asn Arg 225 230 235 240 Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr GluMet Ser Gly Leu Glu 245 250 255 Val Ser Phe Glu Glu Leu Arg Thr Phe GlyGly His Asp Ala Lys Phe 260 265 270 Ile Asp Ser Leu Gln Glu Asn Glu PheArg Leu Tyr Tyr Tyr Asn Lys 275 280 285 Phe Lys Asp Ile Ala Ser Thr LeuAsn Lys Ala Lys Ser Ile Val Gly 290 295 300 Thr Thr Ala Ser Leu Gln TyrMet Lys Asn Val Phe Lys Glu Lys Tyr 305 310 315 320 Leu Leu Ser Glu AspThr Ser Gly Lys Phe Ser Val Asp Lys Leu Lys 325 330 335 Phe Asp Lys LeuTyr Lys Met Leu Thr Glu Ile Tyr Thr Glu Asp Asn 340 345 350 Phe Val LysPhe Phe Lys Val Leu Asn Arg Lys Thr Tyr Leu Asn Phe 355 360 365 Asp LysAla Val Phe Lys Ile Asn Ile Val Pro Lys Val Asn Tyr Thr 370 375 380 IleTyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala Ala Asn Phe 385 390 395400 Asn Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr Lys Leu Lys 405410 415 Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Arg Gly420 425 430 Ile Ile Thr Ser Lys 435 20 441 PRT Clostridium botulinum 20Met Pro Val Thr Ile Asn Asn Phe Asn Tyr Asn Asp Pro Ile Asp Asn 1 5 1015 Asn Asn Ile Ile Met Met Glu Pro Pro Phe Ala Arg Gly Thr Gly Arg 20 2530 Tyr Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Ile Pro Glu 35 4045 Arg Tyr Thr Phe Gly Tyr Lys Pro Glu Asp Phe Asn Lys Ser Ser Gly 50 5560 Ile Phe Asn Arg Asp Val Cys Glu Tyr Tyr Asp Pro Asp Tyr Leu Asn 65 7075 80 Thr Asn Asp Lys Lys Asn Ile Phe Leu Gln Thr Met Ile Lys Leu Phe 8590 95 Asn Arg Ile Lys Ser Lys Pro Leu Gly Glu Lys Leu Leu Glu Met Ile100 105 110 Ile Asn Gly Ile Pro Tyr Leu Gly Asp Arg Arg Val Pro Leu GluGlu 115 120 125 Phe Asn Thr Asn Ile Ala Ser Val Thr Val Asn Lys Leu IleSer Asn 130 135 140 Pro Gly Glu Val Glu Arg Lys Lys Gly Ile Phe Ala AsnLeu Ile Ile 145 150 155 160 Phe Gly Pro Gly Pro Val Leu Asn Glu Asn GluThr Ile Asp Ile Gly 165 170 175 Ile Gln Asn His Phe Ala Ser Arg Glu GlyPhe Gly Gly Ile Met Gln 180 185 190 Met Lys Phe Cys Pro Glu Tyr Val SerVal Phe Asn Asn Val Gln Glu 195 200 205 Asn Lys Gly Ala Ser Ile Phe AsnArg Arg Gly Tyr Phe Ser Asp Pro 210 215 220 Ala Leu Ile Leu Met His GluLeu Ile His Val Leu His Gly Leu Tyr 225 230 235 240 Gly Ile Lys Val AspAsp Leu Pro Ile Val Pro Asn Glu Lys Lys Phe 245 250 255 Phe Met Gln SerThr Asp Ala Ile Gln Ala Glu Glu Leu Tyr Thr Phe 260 265 270 Gly Gly GlnAsp Pro Ser Ile Ile Thr Pro Ser Thr Asp Lys Ser Ile 275 280 285 Tyr AspLys Val Leu Gln Asn Phe Arg Gly Ile Val Asp Arg Leu Asn 290 295 300 LysVal Leu Val Cys Ile Ser Asp Pro Asn Ile Asn Ile Asn Ile Tyr 305 310 315320 Lys Asn Lys Phe Lys Asp Lys Tyr Lys Phe Val Glu Asp Ser Glu Gly 325330 335 Lys Tyr Ser Ile Asp Val Glu Ser Phe Asp Lys Leu Tyr Lys Ser Leu340 345 350 Met Phe Gly Phe Thr Glu Thr Asn Ile Ala Glu Asn Tyr Lys IleLys 355 360 365 Thr Arg Ala Ser Tyr Phe Ser Asp Ser Leu Pro Pro Val LysIle Lys 370 375 380 Asn Leu Leu Asp Asn Glu Ile Tyr Thr Ile Glu Glu GlyPhe Asn Ile 385 390 395 400 Ser Asp Lys Asp Met Glu Lys Glu Tyr Arg GlyGln Asn Lys Ala Ile 405 410 415 Asn Lys Gln Ala Tyr Glu Glu Ile Ser LysGlu His Leu Ala Val Tyr 420 425 430 Lys Ile Gln Met Cys Lys Ser Val Lys435 440

What is claimed is:
 1. A modified neurotoxin comprising: a neurotoxinincluding a structural modification, wherein said structuralmodification is effective to alter a biological persistence of saidmodified neurotoxin relative to an identical neurotoxin without saidstructural modification, and wherein said modified neurotoxin isstructurally different from a naturally existing neurotoxin.
 2. Themodified neurotoxin of claim 1, wherein said structural modification iseffective to enhance a biological persistence of said modifiedneurotoxin.
 3. The modified neurotoxin of claim 1 wherein saidbiological persistence of said modified neurotoxin is reduced relativeto an identical neurotoxin without said structural modification.
 4. Themodified neurotoxin of claim 1 wherein said structural modificationcomprises 1 to about 22 amino acids.
 5. The modified neurotoxin of claim1 wherein said structural modification comprises an amino acid, saidamino acid comprising an R group of 1 to about 12 carbon atoms.
 6. Themodified neurotoxin of claim 1 wherein said structural modificationcomprises a leucine-based motif (SEQ ID NO: 1).
 7. The modifiedneurotoxin of claim 1 wherein said structural modification comprises atyrosine-based motif.
 8. The modified neurotoxin of claim 1 wherein saidstructural modification comprises an amino acid sequence of a botulinumtype A light chain and of an amino acid sequence of a type B lightchain.
 9. The modified neurotoxin of claim 8 wherein said structuralmodification comprises the amino acid sequence KAFK.
 10. The modifiedneurotoxin of claim 8 wherein said structural modification comprises theamino acid sequence YYD in combination with the amino acid sequence YYLin combination with the amino acid sequence T.
 11. The modifiedneurotoxin of claim 1 wherein said structural modification comprises anamino acid derivative.
 12. The modified neurotoxin of claim 1 whereinsaid neurotoxin is selected from the group consisting of botulinum toxintype A, B, C₁, C₂, D, E, F and G.
 13. The modified neurotoxin of claim 1wherein said neurotoxin is botulinum toxin type A.
 14. A modifiedneurotoxin comprising a neurotoxin including a structural modification,wherein said neurotoxin comprises three amino acid sequence regions: a)a first region effective as a cellular binding moiety; b) a secondregion effective to translocate a modified neurotoxin or a part thereofacross an endosome membrane; and c) a third region effective to inhibitexocytosis when released into a cytoplasm of a target cell,

wherein at least one of said first, said second and said third regionsis substantially derived from a Clostridial neurotoxin, said thirdregion includes said structural modification, a modified neurotoxin isstructurally different from a naturally existing neurotoxin, and saidstructural modification is effective to alter a biological persistenceof said modified neurotoxin relative to an identical neurotoxin withoutsaid structural modification.
 15. The modified neurotoxin of claim 14,wherein said neurotoxin is a member selected from a group consisting ofbotulinum toxin serotypes A, B, C₁, C₂, D, E, F, G, tetanus toxin andmixtures thereof.
 16. The modified neurotoxin of claim 14, wherein saidneurotoxin is a member selected from a group consisting of botulinumtoxin serotypes A, B, C₁, C₂, D, E, F and G.
 17. The modified neurotoxinof claim 14, wherein said neurotoxin comprises botulinum toxin serotypesA.
 18. The modified neurotoxin of claim 14, wherein said third region isderived from botulinum toxin serotype A.
 19. The modified neurotoxin ofclaim 14, wherein said third region is not derived from botulinum toxinserotype A.
 20. The modified neurotoxin of claim 14, wherein saidstructural modification includes a biological persistence enhancingcomponent effective to enhance said biological persistence of saidmodified neurotoxin.
 21. The modified neurotoxin of claim 20, whereinsaid biological persistence enhancing component comprises aleucine-based motif of SEQ ID NO:
 1. 22. The modified neurotoxin ofclaim 20, wherein said leucine-based motif comprises a run of sevenamino acids, wherein said run comprises a quintet of amino acids and aduplet of amino acids, wherein said quintet of amino acids defines theamino terminal end of said leucine-based motif and said duplet of aminoacids defines the carboxyl end of said leucine-based motif.
 23. Themodified neurotoxin of claim 22, wherein said quintet of amino acidscomprises an acidic amino acid, wherein said acidic amino acid isselected from a group consisting of a glutamate and an aspartate. 24.The modified neurotoxin of claim 22, wherein said quintet of amino acidscomprises a hydroxyl containing amino acid, wherein said hydroxylcontaining amino acid is selected from the group consisting of a serine,a threonine and a tyrosine.
 25. The modified neurotoxin of claim 24,wherein said hydroxyl containing amino acid can be phophorylated. 26.The modified neurotoxin of claim 22, wherein said duplet of amino acidscomprises at least one amino acid, wherein said amino acid is selectedfrom the group consisting of leucine, isoleucine, methionine, alanine,phenylalanine, tryptophan, valine and tyrosine.
 27. The modifiedneurotoxin of claim 22, wherein said duplet of amino acids is selectedfrom a group consisting of leucine-leucine, leucine-isoleucine,isoleucine-leucine, isoleucine-isoleucine and leucine-methionine. 28.The modified neurotoxin of claim 21, wherein said leucine-based motifcomprises an amino acid sequencephenylalanine-glutamate-phenylalanine-tyrosine-lysine-leucine-leucine ofSEQ ID NO:
 1. 29. A modified neurotoxin wherein said modificationcomprises a tyrosine-based motif.
 30. The modified neurotoxin of claim29 wherein said tyrosine-based motif comprises a run of four aminoacids, wherein an amino acid comprising an N-terminal end of said runcomprises a tyrosine residue and an amino acid comprising a C-terminalend of said run comprises a hydrophobic amino acid.
 31. The modifiedneurotoxin of claim 14, wherein said biological persistence of saidmodified neurotoxin is reduced relative to an identical neurotoxinwithout said structural modification.
 32. The modified neurotoxin ofclaim 31, wherein said structural modification includes a leucine-basedmotif with mutation to one or more amino acids comprising saidleucine-based motif of SEQ ID NO:
 1. 33. The modified neurotoxin ofclaim 31, wherein said structural modification includes a tyrosine-basedmotif with a mutation to one or more amino acids comprising saidtyrosine-based motif.
 34. The modified neurotoxin of claim 31, whereinsaid structural modification comprises an amino acid derivative with amutation to one or more amino acids comprising said amino acidderivative.
 35. A method for enhancing the biological persistence of aneurotoxin of claim 14, wherein a structural modification is fused oradded to said neurotoxin.
 36. The modified neurotoxin of claim 35wherein said structural modification comprises a leucine-based motif.37. The modified neurotoxin of claim 35 wherein said structuralmodification comprises a tyrosine-based motif.
 38. The modifiedneurotoxin of claim 35 wherein said structural modification comprises anamino acid derivative.
 39. A modified neurotoxin comprising: a botulinumtype A neurotoxin including a structural modification, wherein saidstructural modification is effective to alter a biological persistenceof said modified neurotoxin relative to an identical neurotoxin withoutsaid structural modification, wherein said structural modificationcomprises a deletion of amino acids 1 to 8 and 416 to 437 from a lightchain of said neurotoxin.
 40. A modified neurotoxin comprising: abotulinum type A neurotoxin including a structural modification, whereinsaid structural modification is effective to alter a biologicalpersistence of said modified neurotoxin relative to an identicalneurotoxin without said structural modification, wherein said structuralmodification comprises substitution of leucine at position 427 for analanine and leucine at position 428 for an alanine in a light chain ofsaid neurotoxin.
 41. A method for reducing the biological persistence ofa neurotoxin comprising the step of mutating an amino acid of theneurotoxin.
 42. The method of claim 41 which comprises the step ofdeleting or substituting said amino acid of a leucine-based motif withinthe neurotoxin.
 43. The method of claim 41 which comprises the step ofdeleting or substituting said amino acid from a tyrosine-based motifwithin the neurotoxin.
 44. The method of claim 41 which comprises thestep of deleting or substituting an amino acid derivative within theneurotoxin.
 45. A method of treating a condition, comprising a step ofadministering an effective dose of a modified neurotoxin to a mammal totreat a condition, wherein said modified neurotoxin comprises aneurotoxin including a structural modification, and wherein saidstructural modification is effective to alter a biological persistenceof said neurotoxin.
 46. The method of treating a condition of claim 45,wherein said neurotoxin does not comprise a leucine-based motif.
 47. Themethod of treating said condition of claim 46, wherein said structuralmodification includes a biological persistence enhancing component. 48.The method of claim 47 wherein said biological persistence enhancingcomponent comprises a leucine-based motif.
 49. The method of claim 47wherein said biological persistence enhancing component comprises atyrosine-based motif.
 50. The method of claim 47 wherein said biologicalpersistence enhancing component comprises an amino acid derivative. 51.The method of treating said condition of claim 45, wherein saidcondition comprises a condition selected from the group consisting of aneuromuscular disorder, an autonomic disorder and pain.
 52. The methodof treating said condition of claim 51, wherein treatment of saidneuromuscular disorder comprises a step of locally administering aneffective amount of said modified neurotoxin to a muscle or group ofmuscles.
 53. The method of treating said condition of claim 51, whereintreatment of said autonomic disorder comprises a step of locallyadministering an effective amount of said modified neurotoxin to agland.
 54. The method of treating said condition claim 51, whereintreatment of pain comprises a step of administering an effective amountof said modified neurotoxin to a site of pain.
 55. The method oftreating said condition of claim 51, wherein treatment of pain comprisesa step of administering an effective amount of said modified neurotoxinto a spinal cord.
 56. The method of treating said condition of claim 45,wherein said condition is selected from the group consisting ofspasmodic dysphonia, laryngeal dystonia, oromandibular dysphonia,lingual dystonia, cervical dystonia, focal hand dystonia, blepharospasm,strabismus, hemifacial spasm, eyelid disorder, cerebral palsy, focalspasticity, spasmodic colitis, neurogenic bladder, anismus, limbspasticity, tics, tremors, bruxism, anal fissure, achalasia, dysphagia,lacrimation, hyperhydrosis, excessive salivation, excessivegastrointestinal secretions, pain from muscle spasms, headache pain,brow furrows and skin wrinkles.
 57. A modified neurotoxin comprising: aneurotoxin including a structural modification, wherein said structuralmodification is effective to alter a biological activity of saidmodified neurotoxin relative to an identical neurotoxin without saidstructural modification, and wherein said modified neurotoxin isstructurally different from a naturally existing neurotoxin.
 58. Themodified neurotoxin of claim 57, wherein said structural modification iseffective to reduce an exocytosis from a target cell by more than theamount of the exocytosis reduced from the target cell by an identicalneurotoxin without said structural modification.
 59. The modifiedneurotoxin of claim 57, wherein said structural modification iseffective to reduce an exocytosis from a target cell by less than theamount of the exocytosis reduced from the cell by an identicalneurotoxin without said structural modification.
 60. The modifiedneurotoxin of claim 58 or claim 59 wherein the exocytosis is exocytosisof a neurotransmitter.
 61. The modified neurotoxin of claim 57, wherethe modified neurotoxin exhibits an altered biological activity withoutexhibiting an altered biological persistence.
 62. The modifiedneurotoxin of claim 57, where the modified neurotoxin exhibits analtered biological activity and an altered biological persistence. 63.The modified neurotoxin of claim 57, where the modified neurotoxinexhibits an increased biological activity and an increased biologicalpersistence.
 64. The modified neurotoxin of claim 57, where the modifiedneurotoxin exhibits an increased biological activity and a reducedbiological persistence.
 65. The modified neurotoxin of claim 57, wherethe modified neurotoxin exhibits a decreased biological activity and adecreased biological persistence.
 66. The modified neurotoxin of claim57, where the modified neurotoxin exhibits an decreased biologicalactivity and an increased biological persistence.
 67. The modifiedneurotoxin of claim 57 wherein said structural modification comprises aleucine-based motif.
 68. The modified neurotoxin of claim 57, wherein aunit amount of the modified neurotoxin is more efficient to reduce anexocytosis from a cell than is a unit amount of the naturally existingneurotoxin.